RNA-Based Compositions and Methods of Use Thereof

This application is a U.S. National Stage application filed under 35 U.S.C. § 371 of PCT/US2023/073557 filed Sep. 6, 2023, which claims the benefit of priority to U.S. Provisional Patent Application No. 63/403,914, filed Sep. 6, 2022, which are incorporated by reference herein in their entirety.

The sequence listing submitted on Feb. 12, 2026, as an .XML file entitled “11676-003US1_ST26” created on Jan. 7, 2026, and having a file size of 928,518 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

The present disclosure relates to RNA sequences, compositions, and methods of use to prevent viral replication, prevent RNA polymerase activity, activate innate immune responses, or combinations thereof.

Influenza A viruses (IAV) are important human pathogens that generally cause a mild to moderately severe respiratory disease. A range of viral, host, and bacterial factors can influence the outcome of infections with IAV. One important factor is the activation of host protein retinoic acid-inducible gene I (RIG-I) by double-stranded 5′ di- or triphosphorylated RNA. Activated RIG-I translocates to mitochondria, and triggers oligomerization of mitochondrial antiviral signaling protein (MAVS) and subsequent phosphorylation of IRF3 and NF-kB, leading to the expression of innate immune genes, including interferon-β (IFN-β) and IFN-λ. Innate immune gene expression typically leads to a protective antiviral state, but results in an overproduction of cytokines and chemokines when dysregulated. This phenomenon underlies the lethal pathology of infections with 1918 H1N1 pandemic or highly pathogenic avian IAV. Various viral and host factors have been implicated in causing immunopathology, including the products of aberrant viral replication.

The emergence of viral and/or host factors that contribute to immunopathological events within the host, presents the need to develop compositions and methods to prevent viral functions and activate the host immune systems against viral pathogens.

The compositions and methods disclosed herein address these needs.

The present disclosure provides ribonucleic acid (RNA) compositions and methods of use to prevent, reduce, and/or decrease viral replication, increase and/or activate an innate immune response, or a combination thereof to be used for treatment and/or prevention of an infection.

In one aspect, disclosed herein is an engineered RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus.

In some embodiments, the stem portion comprises at least 14 bps in length.

In some embodiments, the target virus comprises a negative-sense RNA virus. In some embodiments, the negative-sense RNA virus comprises Influenza A virus (IAV), Ebola virus, Nipah virus, Hanta virus, Hendra virus, Lassa virus, or Rabies virus.

In some embodiments, the RNA sequence comprises between about 40 to about 80 nucleotides. In some embodiments, the RNA sequence comprises at least 60% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 70% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 80% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 90% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises any one of sequences selected from SEQ ID NO: 4-30.

In some embodiments, the footprint of the RNA polymerase comprises an area within the RNA polymerase capable of holding a designated number of nucleotides. In some embodiments, the designated number of nucleotides comprises about 20 nucleotides. In some embodiments, the 5′ promoter comprises about 12 nucleotides in length. In some embodiments, the 3′ promoter comprises about 13 nucleotides in length.

In one aspect, disclosed herein is a cell expressing the engineered RNA of any preceding aspect.

In one aspect, disclosed herein is a method of reducing viral replication, inducing activation of an innate immune response to a target virus, or a combination thereof, the method comprising engineering an RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus, and contacting the RNA sequence to the RNA polymerase of the target virus, wherein the RNA sequence forms a template loop (t-loop) around the RNA polymerase to reduce viral replication, and wherein the RNA sequence comprises an agonist to activate the innate immune response.

In one aspect, disclosed herein is a method of preventing RNA polymerase activity, the method comprising engineering an RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 14 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus, and contacting the RNA sequence to the RNA polymerase of the target virus, wherein the RNA sequence forms a template loop (t-loop) around the RNA polymerase to stop RNA polymerase activity.

In one aspect, disclosed herein is a method of treating and/or preventing a viral infection, the method comprising engineering an RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus, and administering the RNA sequence to a subject, wherein the RNA sequence contacts the RNA polymerase of the target virus, and wherein the RNA sequence reduces viral replication and/or activates the innate immune response in the subject relative to an untreated control subject.

In one aspect, disclosed herein is a method of performing a template loop (t-loop) analysis, the method comprising identifying a template loop RNA sequence, blocking off a portion of the template loop RNA sequence to represent a footprint of a virus RNA polymerase, determining a t-loop ΔG, an upstream ΔG for a stretch of 10 nucleotides of the footprint, and a downstream ΔG for a stretch of 10 nucleotides of the footprint, and determining a ΔΔG for a likelihood of t-loop formation by subtracting the upstream ΔG and downstream ΔG from the t-loop ΔG, and moving the footprint in a one-nucleotide increment along the template loop RNA sequence, and repeating step c) until the ΔΔG values have been determined for an entire template loop RNA sequence.

In some embodiments, the stem portion comprises at least 14 bps in length.

In some embodiments, the target virus comprises a negative-sense RNA virus. In some embodiments, the negative-sense RNA virus comprises Influenza A virus (IAV), Ebola virus, Nipah virus, Hanta virus, Hendra virus, Lassa virus, or Rabies virus.

In some embodiments, the RNA sequence comprises between about 40 to about 80 nucleotides. In some embodiments, the RNA sequence comprises any one of sequences selected from SEQ ID NO: 4-30.

In some embodiments, the footprint of the RNA polymerase comprises an area within the RNA polymerase capable of holding a designated number of nucleotides. In some embodiments, the designated number of nucleotides comprises about 20 nucleotides. In some embodiments, the 5′ promoter comprises about 12 nucleotides in length. In some embodiments, the 3′ promoter comprises about 13 nucleotides in length.

In some embodiments, the stem-loop inhibits the RNA polymerase from replicating a genome of the target virus. In some embodiments, the RNA sequence forms a template loop (t-loop) around the RNA polymerase to reduce viral replication.

In some embodiments, the innate immune response comprises binding a host pathogen receptor to the RNA sequence. In some embodiments, the host pathogen receptor comprises a retinoic acid-inducible gene I (RIG-I).

In one aspect, disclosed herein is a non-transitory computer-readable storage medium comprising instructions that, when executed, cause at least one processor to perform the method of any preceding aspect.

In one aspect, disclosed herein is a computer-implemented method for generating at least one optimal ribonucleic acid (RNA) sequence for reducing viral replication and/or inducing activation of an innate response to a target virus, the computer-implemented method comprising retrieving, by one or more processors, an RNA polymerase of the target virus, performing, by the one or more processors, a template loop (t-loop) analysis operation on the RNA polymerase, determining, by the one or more processors, at least one optimal RNA sequence corresponding with the RNA polymerase of the target virus based on results of the t-loop analysis operation, and outputting, by the one or more processors, a ΔG, a location of the ΔG, a t-loop structure, or combinations thereof. In some embodiments, the output comprises the at least one optimal RNA sequence.

In some embodiments, the computer-implemented method further comprises receiving, by the one or more processors, one or more user-defined parameters associated with the RNA polymerase of the target virus, and performing, by the one or more processors, the t-loop analysis operation based at least on the one or more user-defined parameters. In some embodiments, the t-loop analysis operation is used interchangeably with a sliding window operation. In some embodiments, the computer-implemented method further comprises determining, by the one or more processors, at least one criterion of a composition for administration to a subject based on the at least one optimal RNA sequence.

The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

The following definitions are provided for the full understanding of terms used in this specification.

The terms “about” and “approximately” are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

“Comprising” is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. “Consisting essentially of” when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% or more increase so long as the increase is statistically significant.

A “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

“Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., viral replication or viral infection). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces viral infection” or “reduces viral replication” means reducing the rate viral infection or rate of viral replication relative to a standard or a control.

By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of infection), during early onset (e.g., upon initial signs and symptoms of infection), or after an established development of infection.

The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

A “promoter,” as used herein, refers to a sequence in DNA that mediates the initiation of transcription by an RNA polymerase. Transcriptional promoters may comprise one or more of a number of different sequence elements as follows: 1) sequence elements present at the site of transcription initiation; 2) sequence elements present upstream of the transcription initiation site and; 3) sequence elements down-stream of the transcription initiation site. The individual sequence elements function as sites on the DNA, where RNA polymerases and transcription factors facilitate positioning of RNA polymerases on the DNA bind.

“Expression” as used herein refers to the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce a peptide/protein end product, and ultimately affect a phenotype, as the final effect.

As used herein, the term “polymerase” refers to an enzyme that synthesizes long chains of polymers or nucleic acids. RNA polymerases are used to assemble RNA molecules, respectively, by copying a nucleic acid template strand using base-pairing interactions.

The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

“Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disease or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disease or other undesirable physiological condition (including, but not limited to Influenza). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some aspects, the composition disclosed herein comprises an engineered RNA sequence and a pharmaceutically effective carrier.

“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.

Remington's Pharmaceutical Sciences, As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g.,21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of DNA nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule.

A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material. Herein the terms “nucleic acid” and “polynucleotide” are used interchangeably throughout the disclosure.

The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

A “genome” refers to a complete set of genes or genetic material present within a cell, tissue, or organism, including, but not limited to pathogenic genomes (i.e.: viral genomes).

Variants comprising a fragment of a reference nucleotide sequence are contemplated herein. A “fragment” is a portion of a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides of a reference polynucleotide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides of a reference polynucleotide. Fragments may be preferentially selected from certain regions of a molecule, for example the 5′-terminal region and/or the 3′ terminal region of a polynucleotide. The term “at least a fragment” encompasses the full-length polynucleotide.

A “host” refers to any animal (either vertebrate or invertebrate) or plant that harbors a smaller organism; whether their relationship is parasitic, pathogenic, or symbiotic, where the smaller organism generally uses the animal or plant for shelter and/or nourishment. The smaller organism can be a microorganism, such as bacteria, viruses, fungi, a parasite, including, but not limited to worms and insects.

“Serotype” as used herein refers to a distinct variation within a species of bacteria or virus or among immune cells of different individuals. These microorganisms, viruses, or cells are classified together based on their surface antigens, allowing the epidemiologic classification of organisms to the subspecies level.

As used herein, the term infection refers to the invasion of tissues by pathogens, their multiplication, and reaction of host tissues to the infectious agent and any toxins they release. Infections can be caused by a wide range of pathogen, most common are viruses.

As used herein, “downstream” refers to the relative position of a genetic sequence, either DNA or RNA. Downstream relates to the 5′ to 3′ direction relative to the start site of transcription, wherein downstream is usually closer to the 3′ end of a genetic sequence.

As used herein, “upstream” refers to the relative position of a genetic sequence, either DNA or RNA. Upstream relates to the 5′ to 3′ directions relative to the start site of transcription, wherein upstream is usually closer to the 5′ end of a genetic sequence.

A “virus” is a microscopic infectious agent that replicates only inside the living cells of an organism. Viruses can infect all life forms, including mammalian and non-mammalian animals, plants, and other microorganisms. A complete virus, also known as a virion, consists of nucleic acid genetic material surrounded by a protective coat of protein called a capsid. Virus can have a lipid envelope derived from the infected host cell membrane. In general, there are five morphological virus types including helical, icosahedral, prolate, enveloped, and complex virus. A virus can either have a DNA or RNA genome, though a vast majority have RNA genomes. Irrespective of the type of nucleic acid genome, a viral genome can be either a single-stranded genome or a double-stranded genome.

RNA viruses are a group of viruses that have ribonucleic acid (RNA), in the form of single stranded RNA or double stranded RNA, as its genetic material. RNA viruses can be classified according to the polarity of the RNA into negative-sense RNA viruses or positive-sense RNA viruses. Viral RNA from a negative-sense RNA virus is complementary to messenger RNA (mRNA), and thus must be converted to positive-sense RNA by an RNA-dependent RNA polymerase before translation into viral proteins. Positive-sense viral RNA, on the other hand, is identical to mRNA, and can thus be translated immediately.

RNA viruses generally comprise very high mutation rates because viral RNA polymerases lack the proofreading functions of DNA polymerases. This contributes to a genetic diversity of RNA molecules (viral RNA or vRNA) produced by RNA viruses. Viral RNA polymerases are also shown to generate aberrant RNA molecules called mini viral RNAs (mvRNAs) during replication of the viral genome. Given the genetic diversity of RNA viruses, there is a need to generate RNA sequences of compositions to prevent RNA virus replication in a host.

In one aspect, disclosed herein is an engineered RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus.

In one aspect, disclosed herein is an engineered RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of influenza A virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the influenza A virus.

In some embodiments, the RNA sequences disclosed herein comprise or match a footprint of an RNA polymerase from a target virus. The footprint refers to an area or region within the RNA polymerase that encloses or surrounds an RNA molecule. The footprint also allows for a designated or optimal number of nucleotides to be held within the RNA polymerase. The footprint includes the entry and exit pathways for the RNA molecule to enter upon conversion to positive-sense RNA and exit after conversion to positive-sense RNA.

In some embodiments, the RNA sequences disclosed herein are suitable for RNA polymerases from a negative-sense RNA virus. The RNA sequences disclosed herein traps the viral replication complex (RNA polymerase) or competes with endogenous vRNAs to inhibit viral replication. The RNA sequences disclosed herein are potent inhibitors of viral infections including, but not limited to infections caused by influenza A virus, influenza B virus, influenza C virus, Ebola virus, Nipah virus, Hanta virus, Hendra virus, Lassa virus, or Rabies virus. Thus, such viruses are sensitive to template loops (t-loops) and are inhibited in similar manners. The RNA sequences disclosed herein can be coupled to a ubiquitin ligase recognition signal (e.g., PROTAC) and trigger the degradation of viral proteins by the proteasome.

In some embodiments, the RNA sequences disclosed herein can activate the immune response upon viral infection and can induce a protective antiviral response. There are various previous methods on using defective interfering RNA viruses/particles, but these rely on using natural sequences. The sequences and the algorithm that are disclosed herein engineer viruses capable of inducing a protective response more robustly, for instance by inserting the sequences into the genome or creating viruses in which one of the viral genome segments is replaced with an RNA capable of trapping the viral replication complex. The RNA sequences disclosed herein can be added to live-attenuated vaccines and act as an adjuvant. In the vaccines, the RNA sequence(s) is bound by the live virus upon infection. After initial steps of infection, the RNA sequences limit viral replication and/or trigger a protective immune response. Said vaccines can be administered during an outbreak of a known or novel virus to provide subjects with a protective immune response before traditional or RNA vaccines are available. Said vaccine can also be administered to healthcare workers before exposure to viruses.

In some embodiments, disclosed herein are RNA sequences used to inhibit influenza virus replication and trigger activation of the innate immune system while the RNA is bound by the viral replication complex (RNA polymerase). The template loop (t-loop) portion of the RNA sequence inhibits other negative viruses, not limited to influenza viruses. Thus, the RNA sequences disclosed herein inhibit viral RNA polymerase activity and/or viral replication in RNA viruses that are similar in size and work mechanistically in similar ways to influenza viruses and other negative-sense RNA viruses.

Under appropriate parameters, the RNA sequence(s) hybridizes, or binds internally to form the t-loop. To reduce viral replication and induce activation of the innate immune response, the stem of the t-loop comprises at least 5 bp long. To prevent the activity of the RNA polymerase entirely, the stem comprises at least 14 bp long. The t-loop is flanked by the natural influenza A virus 5′ and 3′ promoter RNA sequences, which are 12 and 13 nt respectively.

In some embodiments, the RNA sequence can reduce or completely stop the activity of the RNA polymerase.

In some embodiments, the stem portion comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 bps in length. In some embodiments, the loop portion comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length.

In some embodiments, the target virus comprises a negative-sense RNA virus. In some embodiments, the negative-sense RNA virus comprises Influenza A virus (IAV), Ebola virus, Nipah virus, Hanta virus, Hendra virus, Lassa virus, or Rabies virus. It should be understood that negative-sense viruses can comprise multiple types of genomes ranging from a single RNA molecule up to eight segments of RNA polymers. For example, IAV and Influenza V virus (IBV) comprise eight segments, while Influenza C virus comprises seven segments. Thus, in some embodiments the engineered RNA sequence targets at least one single RNA molecule or targets up to 8 segments of RNA.

In some embodiments, the RNA sequence comprises between about 40 to about 130 nucleotides. In some embodiments, the RNA sequence comprises between about 52 to about 71 nucleotides. In some embodiments, the RNA sequence comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 nucleotides. It should be noted that the RNA sequences disclosed herein are shorter than defective interfering influenza virus RNAs (including, but not limited to defective interfering (DI) RNAs or DI viruses) described in the art.

In some embodiments, the RNA sequence comprises at least 60% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 70% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 80% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 90% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 95% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises at least 99% sequence identity to any one of SEQ ID NO: 4-30. In some embodiments, the RNA sequence comprises any one of sequences selected from SEQ ID NO: 4-30.

In some embodiments, the RNA sequence comprises SEQ ID NO: 4, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 5, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 6, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 7, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 8, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 9, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 10, or a variant thereof.

In some embodiments, the RNA sequence comprises SEQ ID NO: 11, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 12, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 13, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 14, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 15, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 16, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 17, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 18, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 19, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 20, or a variant thereof.

In some embodiments, the RNA sequence comprises at least 60% sequence identity to any one of SEQ ID NO: 13. In some embodiments, the RNA sequence comprises at least 70% sequence identity to any one of SEQ ID NO: 13. In some embodiments, the RNA sequence comprises at least 80% sequence identity to any one of SEQ ID NO: 13. In some embodiments, the RNA sequence comprises at least 90% sequence identity to any one of SEQ ID NO: 13. In some embodiments, the RNA sequence comprises at least 95% sequence identity to any one of SEQ ID NO: 13. In some embodiments, the RNA sequence comprises at least 99% sequence identity to any one of SEQ ID NO: 13.

In some embodiments, the RNA sequence comprises at least 60% sequence identity to any one of SEQ ID NO: 14. In some embodiments, the RNA sequence comprises at least 70% sequence identity to any one of SEQ ID NO: 14. In some embodiments, the RNA sequence comprises at least 80% sequence identity to any one of SEQ ID NO: 14. In some embodiments, the RNA sequence comprises at least 90% sequence identity to any one of SEQ ID NO: 14. In some embodiments, the RNA sequence comprises at least 95% sequence identity to any one of SEQ ID NO: 14. In some embodiments, the RNA sequence comprises at least 99% sequence identity to any one of SEQ ID NO: 14.

In some embodiments, the RNA sequence comprises SEQ ID NO: 21, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 22, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 23, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 24, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 25, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 26, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 27, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 28, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 29, or a variant thereof. In some embodiments, the RNA sequence comprises SEQ ID NO: 30, or a variant thereof.

In some embodiments, the RNA sequence comprises at least 60% sequence identity to any one of SEQ ID NO: 25. In some embodiments, the RNA sequence comprises at least 70% sequence identity to any one of SEQ ID NO: 25. In some embodiments, the RNA sequence comprises at least 80% sequence identity to any one of SEQ ID NO: 25. In some embodiments, the RNA sequence comprises at least 90% sequence identity to any one of SEQ ID NO: 25. In some embodiments, the RNA sequence comprises at least 95% sequence identity to any one of SEQ ID NO: 25. In some embodiments, the RNA sequence comprises at least 99% sequence identity to any one of SEQ ID NO: 25.

In some embodiments, SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3 can be used as a control sequence (such as, for example a negative control or a positive control).

In some embodiments, the footprint of the RNA polymerase comprises an area within the RNA polymerase capable of holding a designated number of nucleotides. In some embodiments, the designated number of nucleotides comprises about 20 nucleotides. In some embodiments, the designated number of nucleotides comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.

In some embodiments, the 5′ promoter comprises about 12 nucleotides in length. In some embodiments, the 5′ promoter comprises about 10, 11, 12, 13, 14, 15 nucleotides in length. In some embodiments, the 3′ promoter comprises about 13 nucleotides in length. In some embodiments, the 3′ promoter comprises about 10, 11, 12, 13, 14, 15 nucleotides in length.

In one aspect, disclosed herein is a cell expressing the engineered RNA of any preceding aspect. In some embodiments, the cell expresses or comprises a vector encoding the engineered RNA.

In some embodiments, the cell is a mammalian cell or a bacterial cell.

It should be understood that any vector capable of stably expressing the engineered RNA sequences can be used. The word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed or transduced into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In some embodiments, the vector comprises a nucleic construct containing a nucleotide sequence which is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable RNA ribosome binding sites, and sequences which control the termination of transcription and translation.

In some embodiments, the vector comprises a lipid nanoparticle. Lipid nanoparticles can be used to deliver engineered RNA sequences to a cell. In some embodiments, the RNA sequence can be introduced into a host cell through genetic modification. In some embodiments, the RNA sequence is introduced into a host cell by any DNA or RNA delivery technology known in the art, or combinations thereof.

In some embodiments, the vector comprises a plasmid. In some embodiments, the vector comprises an RNA polymerase I (pol I) promoter. In some embodiments, the vector comprises flanking the RNA sequence with a hepatitis delta virus ribozyme sequence. A non-limiting example of expression of an IAV is described in Fodor et al. “Rescue of Influenza A Virus from Recombinant DNA”. Journal of Virology. November 1999 pg. 9679-9682, which is incorporated herein in its entirety as a reference for its teaching of designing a vector (such as, for example a plasmid) comprising IAV genes and expressing said vector in cells.

Methods of Treating, Preventing, Reducing, and/or Decreasing Viral Infections

Viral replication refers to the formation process of biological viruses during an infection in host cells. Because viruses can only multiply within a living host cell, the host cell must supply the energy, replicative machinery, and the low molecular weight precursors for synthesis of viral proteins and nucleic acids. The process of viral replication occurs in seven stages, including: 1) Attachment, wherein the virus attaches to the cell membrane of the host cell and injects genetic material into the host to initiate infection; 2) Entry, wherein the host cell membrane invaginates, or internalizes, the virus particles; 3) Uncoating, wherein the enzymes from the host cell strips away the virus protein coat to expose viral genome; 4) Transcription, wherein viral RNA can be directly translated into viral protein (positive-sense RNA viruses), or must first be transcribed into positive-sense RNA before protein translation (negative-sense or DNA viruses); 5) Synthesis of viral components, wherein the components of the virus are manufactured using existing enzymes and organelles of the host; 6) Viral assembly, wherein the newly synthesized genome and proteins are assembled into a new, active virus; and 7) Release, wherein the newly assembled viruses are released by sudden rupture of the host cell or gradual extrusion of viruses from the host cell. It should be noted that during step 4, negative-sense RNA polymerases can generate aberrant RNA sequences (vRNAs and/or mvRNAs).

It has been contemplated that generation of mvRNAs trigger an innate immune response against viral infections. The innate immune response refers to the first line of defense against invading pathogens that includes immune systems cells and proteins that protect against pathogens that have entered the host body. “The innate and adaptive immune systems” is incorporate herein by reference, in its entirety, for the teachings of the components and functions of the innate immune system (InformedHealth.org. Cologne, Germany: Institute for Quality and Efficiency in Health Care (IQWiG); 2006—. The innate and adaptive immune systems. Available from: www.ncbi.nlm.nih.gov/books/NBI279396). A non-limiting example of an innate immune response includes the retinoic acid-inducible gene I (RIG-I) that is an RNA sensor important for detecting viral infections. The RIG-I sensor is a host pathogen receptor that, upon binding a target RNA molecule, initiates a cascade of signaling pathways that leads to interferon (IFN) expression. This innate response causes release of IFN proteins that activate and allow communication between immune system cells to eradicate the infectious pathogen.

The present disclosure provides methods that manipulate and/or optimize the above mentioned processes to prevent, reduce, and/or decrease viral replication; prevent, reduce, and/or decrease RNA polymerase activity; activate and/or increase an innate immune response; treat and/or prevent a viral infection; or any combinations thereof.

In one aspect, disclosed herein is a method of reducing viral replication, inducing activation of an innate immune response to a target virus, or a combination thereof, the method comprising engineering an RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus, and contacting the RNA sequence to the RNA polymerase of the target virus, wherein the RNA sequence forms a template loop (t-loop) around the RNA polymerase to reduce viral replication, and wherein the RNA sequence comprises an agonist to activate the innate immune response.

In one aspect, disclosed herein is a method of preventing RNA polymerase activity, the method comprising engineering an RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 14 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus, and contacting the RNA sequence to the RNA polymerase of the target virus, wherein the RNA sequence forms a template loop (t-loop) around the RNA polymerase to stop RNA polymerase activity.

In one aspect, disclosed herein is a method of treating and/or preventing a viral infection, the method comprising engineering an RNA sequence comprising a stem-loop, wherein the stem-loop comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, wherein the loop portion matches a footprint of an RNA polymerase of a target virus, and wherein the stem loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus, and administering the RNA sequence to a subject, wherein the RNA sequence contacts the RNA polymerase of the target virus, and wherein the RNA sequence reduces viral replication and activates the innate immune response in the subject relative to an untreated control subject.

Disclosed herein are methods to activate the innate immune response using RNA-based inhibition of influenza virus replication.

In some embodiments, the method comprises a stem portion with 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 bps in length. In some embodiments, the method comprises a loop portion with about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length.

In some embodiments, the method comprises an RNA sequence with between about 40 to about 80 nucleotides. In some embodiments, the method comprises an RNA sequence with between about 52 to about 71 nucleotides. In some embodiments, the method comprises an RNA sequence with about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 nucleotides.

In some embodiments, the target virus comprises a negative-sense RNA virus. In some embodiments, the negative-sense RNA virus comprises Influenza A virus (IAV), Ebola virus, Nipah virus, Hanta virus, Hendra virus, Lassa virus, or Rabies virus.

In some embodiments, the method comprises any one of sequences selected from SEQ ID NO: 4-30.

In some embodiments, the footprint of the RNA polymerase comprises an area within the RNA polymerase capable of holding a designated number of nucleotides. In some embodiments, the designated number of nucleotides comprises about 20 nucleotides. In some embodiments, the designated number of nucleotides comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.

In some embodiments, the method comprises a 5′ promoter with about 12 nucleotides in length. In some embodiments, the method comprises a 5′ promoter with about 10, 11, 12, 13, 14, 15 nucleotides in length. In some embodiments, the method comprises a 3′ promoter with about 13 nucleotides in length. In some embodiments, the method comprises a 3′ promoter with about 10, 11, 12, 13, 14, 15 nucleotides in length.

In some embodiments, the stem-loop inhibits the RNA polymerase from replicating a genome of the target virus. In some embodiments, the stem-loop inhibits the RNA polymerase from replicating negative-sense RNA. In some embodiments, the RNA sequence forms a template loop (t-loop) around the RNA polymerase to reduce viral replication. In some embodiments, the method inhibits transcription, synthesis, assembly, and/or release of viral components.

In some embodiments, the innate immune response comprises binding a host pathogen receptor to the RNA sequence. In some embodiments, the host pathogen receptor comprises a retinoic acid-inducible gene I (RIG-I). In some embodiments, the method comprises expression and/or release of IFN proteins against the viral infection.

2 FIG.A nd Template loop (t-loop) structures are RNA sequences that form a nucleic acid complex around an RNA polymerase to stall or prevent replication. Under optimized parameters, the t-loop can also initiate an innate immune response (such as, for example triggering a RIG-I RNA sensor). To optimally prevent RNA polymerase functions, such as, for example replication, the t-loop matches or comprises a footprint of the RNA polymerase from a target virus, such as, for example, influenza A virus. A matching footprint allows the RNA sequence to reside within the entry, exit, and interior channels of the RNA polymerase (See(2image; T-loop RNA structure)). The t-loop structure forms around the RNA polymerase when the 3′ terminus of the RNA sequence (generally located at the exit channel of the RNA polymerase) can hybridize, or bind, to the upstream 5′ terminus of the RNA sequence (generally located at the entry channel of the RNA sequence). The hybridizing, or binding, of the 3′ terminus to the 5′ terminus causes the internal nucleotides of the RNA sequence to loop around and through the RNA polymerase. The formation of at least one t-loop around an RNA polymerase can stall the RNA polymerase from replicating the viral genome. It should be understood that RNA sequence can form 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more t-loops around and through the RNA polymerase.

Depending on the purpose of the RNA sequence (inhibition, innate immune activation, or both), the stem of a template loop (t-loop) can be modified, and the overall length of the RNA sequence optimized. Since the mechanism of action of t-loops is well-defined, an algorithm was designed to evaluate and optimize the sequences in silico. Additional t-loop sequences, aside from those sequences disclosed herein, can be quickly identified using the in silico analyses disclosed herein. The specific parameters can be defined to broadly claim the whole RNA sequence space that yields t-loop sequences with similar properties. Thus, the present disclosure provides methods of analyzing t-loop sequences for use in inhibiting RNA polymerase activity, inhibiting viral replication, activating an innate immune response, or combinations thereof.

In some embodiments, the analysis includes detecting or identifying the free energy of the t-loop, the upstream sequence, and downstream sequence of an RNA sequence template. In some embodiments, the analysis includes determining the differences in free energies between the t-loop, the upstream sequence, and the downstream sequence of the RNA sequence template.

Free energy, also referred to as ΔG, refers to a thermodynamic property that defines an energy property or function of a system in thermodynamic equilibrium. Free energy has the dimensions of energy, and its value is determined by the state of the system. Free energy is used to determine how systems change and how much work (in the form of energy) they can produce. When referring to nucleic acid systems, nucleic acid free energy refers to how external and internal factors (such as, for example temperature, the number of nucleotides, and/or the type of nucleotides) affect the formation and/or binding of secondary or tertiary nucleic acid structures (including, but not limited to hairpins, stem loops, and/or t-loops). Detection of a ΔG for a given RNA sequence template informs on the parameters needed to optimally form a t-loop structure. Such parameters include, but are not limited to the appropriate temperature, the optimal number of nucleotides, and/or the optimal positioning of adenine, thymine, cytosine, and guanine nucleotides for hybridization of the RNA sequence template.

In some embodiments, the temperature parameter for forming t-loops ranges from about 35° C. to about 40° C. In some embodiments, the temperature parameter for forming t-loops ranges from about 36° C. to about 38° C. In some embodiments, the temperature parameter for forming t-loops comprises about 37° C. In some embodiments, the temperature parameter for forming t-loops comprises about 36.0° C., 36.1° C., 36.2° C., 36.3° C., 36.4° C., 36.5° C., 36.6° C., 36.7° C., 36.8° C., 36.9° C., 37.0° C., 37.1° C., 37.2° C., 37.3° C., 37.4° C., 37.5° C., 37.6° C., 37.7° C., 37.8° C., 37.9° C., 38.0° C., 38.1° C., 38.2° C., 38.3° C., 38.4° C., 38.5° C., 38.6° C., 38.7° C., 38.8° C., 38.9° C., or 39.0° C.

Additional optimization is required by determining the differences in free energies (ΔΔG) between the ΔG of the t-loop, ΔG of the upstream (5′) terminus sequence, and ΔG of the downstream (3′) terminus sequence (or promoters) of the RNA sequence template. Determining the ΔΔG can be repeat as many times as necessary until the ΔΔG values have been determined for an entire template loop RNA sequence.

In one aspect, disclosed herein is a method of performing a t-loop analysis, the method comprising identifying a template loop RNA sequence, blocking off a portion of the template loop RNA sequence to represent a footprint of a virus RNA polymerase, determining a t-loop ΔG, an upstream ΔG for a stretch of at 10 nucleotides of the footprint, and a downstream ΔG for a stretch of at 10 nucleotides of the footprint, and determining a ΔΔG for a likelihood of t-loop formation by subtracting the upstream ΔG and downstream ΔG from the t-loop ΔG, and moving the footprint in a one-nucleotide increment along the template loop RNA sequence, and repeating the previous step until the ΔΔG values have been determined for an entire template loop RNA sequence.

In one aspect, disclosed herein is a method of designing single stranded RNA molecules that regulate viral replication and stimulate activation of the innate immune response leading to the production of interferons. In some embodiments, the method regulates influenza A viral replication.

In some embodiments, the stretch of nucleotides comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of the footprint.

Disclosed herein is an algorithm (Python code) to test the design of new RNA sequences in silico.

18 FIG. It should be appreciated that the logical operations described herein with respect to the various figures may be implemented (1) as a sequence of computer-implemented acts or program modules (i.e., software) running on a computing device (e.g., the computing device described in), (2) as interconnected machine logic circuits or circuit modules (i.e., hardware) within the computing device and/or (3) a combination of software and hardware of the computing device. Thus, the logical operations discussed herein are not limited to any specific combination of hardware and software. The implementation is a matter of choice dependent on the performance and other requirements of the computing device. Accordingly, the logical operations described herein are referred to variously as operations, structural devices, acts, or modules. These operations, structural devices, acts and modules may be implemented in software, in firmware, in special-purpose digital logic, and any combination thereof. It should also be appreciated that more or fewer operations may be performed than shown in the figures and described herein. These operations may also be performed in a different order than those described herein.

18 FIG. 1800 1800 1800 Referring to, an example computing deviceupon which embodiments of the invention may be implemented is illustrated. It should be understood that the example computing deviceis only one example of a suitable computing environment upon which embodiments of the invention may be implemented. Optionally, the computing devicecan be a well-known computing system including, but not limited to, personal computers, servers, handheld or laptop devices, multiprocessor systems, microprocessor-based systems, personal network computers (PCs), minicomputers, mainframe computers, embedded systems, and/or distributed computing environments including a plurality of any of the above systems or devices. Distributed computing environments enable remote computing devices, which are connected to a communication network or other data transmission medium, to perform various tasks. In the distributed computing environment, the program modules, applications, and other data may be stored on local and/or remote computer storage media.

1800 1806 1804 1804 1802 1806 1800 1800 1800 18 FIG. In its most basic configuration, the computing devicetypically includes at least one processing unitand system memory. Depending on the exact configuration and type of computing device, system memorymay be volatile (such as random-access memory (RAM)), non-volatile (such as read-only memory (ROM), flash memory, etc.), or some combination of the two. This most basic configuration is illustrated inby the dashed line. The processing unitmay be a standard programmable processor that performs arithmetic and logic operations necessary for the operation of the computing device. The computing devicemay also include a bus or other communication mechanism for communicating information among various components of the computing device.

1800 1800 1808 1810 1800 1816 1800 1814 1812 1800 Computing devicemay have additional features/functionality. For example, the computing devicemay include additional storage such as removable storageand non-removable storageincluding, but not limited to magnetic or optical disks or tapes. Computing devicemay also contain network connection(s)that allow the device to communicate with other devices. Computing devicemay also have input device(s)such as a keyboard, mouse, touch screen, etc. Output device(s), such as a display, speakers, printer, etc., may also be included. The additional devices may be connected to the bus in order to facilitate communication of data among the components of the computing device. All these devices are well-known in the art and need not be discussed at length here.

1806 1800 1806 1804 1808 1810 The processing unitmay be configured to execute program code encoded in tangible, computer-readable media. Tangible, computer-readable media refers to any media that is capable of providing data that causes the computing device(i.e., a machine) to operate in a particular fashion. Various computer-readable media may be utilized to provide instructions to the processing unitfor execution. Example of tangible, computer-readable media may include but is not limited to, volatile media, non-volatile media, removable media and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data. System Memory, removable storage, and non-removable Storageare all examples of tangible computer storage media. Examples of tangible, computer-readable recording media include but are not limited to, an integrated circuit (e.g., field-programmable gate array or application-specific IC), a hard disk, an optical disk, a magneto-optical disk, a floppy disk, a magnetic tape, a holographic storage medium, a solid-state device, RAM, ROM, electrically erasable program read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices.

1806 1804 1804 1806 1804 1808 1810 1806 In an example implementation, the processing unitmay execute program code stored in the system memory. For example, the bus may carry data to the system memory, from which the processing unitreceives and executes instructions. The data received by the system memorymay optionally be stored on the removable storageor the non-removable storagebefore or after execution by the processing unit.

It should be understood that the various techniques described herein may be implemented in connection with hardware or software or, where appropriate, with a combination thereof. Thus, the methods and apparatuses of the presently disclosed subject matter, or certain aspects or portions thereof, may take the form of program code (i.e., instructions) embodied in tangible media, such as floppy diskettes, CD-ROMs, hard drives, or any other machine-readable storage medium wherein, when the program code is loaded into and executed by a machine, such as a computing device, the machine becomes an apparatus for practicing the presently disclosed subject matter. In the case of program code execution on programmable computers, the computing device generally includes a processor, a storage medium readable by the processor (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. One or more programs may implement or utilize the processes described in connection with the presently disclosed subject matter, for example, through the use of an application programming interface (API), reusable controls, or the like. Such programs may be implemented in a high-level procedural or object-oriented programming language to communicate with a computer system. However, the program(s) can be implemented in assembly or machine language if desired. In any case, the language may be a compiled or interpreted language, and it may be combined with hardware implementations.

In one aspect, disclosed herein is a non-transitory computer-readable storage medium comprising instructions that, when executed, cause at least one processor to perform the method of any preceding aspect.

In some implementations, the techniques described herein relate to a computer-implemented method for generating at least one optimal RNA sequence for reducing viral replication and/or inducing activation of an innate response to a target virus, the computer-implemented method including: retrieving, by one or more processors, an RNA polymerase of the target virus; performing, by the one or more processors, a template loop (t-loop) analysis operation on the RNA polymerase; determining, by the one or more processors and based on results from the t-loop analysis operation and/or sliding window operation, at least one optimal RNA sequence corresponding with the RNA polymerase of the target virus; and outputting, by the one or more processors, the at least one optimal RNA sequence (e.g., via a display device or other output device).

In some implementations, the techniques described herein relate to a computer-implemented method, further including receiving, by the one or more processors, one or more user-defined parameters associated with the RNA polymerase of the target virus; and performing, by the one or more processors, the t-loop analysis operation based at least on the one or more user-defined parameters.

In some implementations, the techniques described herein relate to a computer-implemented method, further including determining, by the one or more processors, at least one criterion of a composition for administration to a subject based on the at least one optimal RNA sequence.

19 FIG. 18 FIG. 1900 1900 1800 is a flowchart of an example computer-implemented methodfor generating at least one optimal RNA sequence for reducing viral replication and/or inducing activation of an innate response to a target virus. In some implementations, the methodcan be performed by a processing circuitry (for example, but not limited to, an application-specific integrated circuit (ASIC), or a central processing unit (CPU)). In some examples, the processing circuitry may be electrically coupled to and/or in electronic communication with other circuitries of an example computing device, such as, but not limited to, the example computing devicedescribed above in connection with. In some examples, embodiments may take the form of a computer program product on a non-transitory computer-readable storage medium storing computer-readable program instruction (e.g., computer software). Any suitable computer-readable storage medium may be utilized, including non-transitory hard disks, CD-ROMs, flash memory, optical storage devices, or magnetic storage devices.

1910 1800 1910 At step/operation, at least one processor (such as, but not limited to, at least one processor or processing circuitry of the computing device) retrieves an RNA polymerase of the target virus. In some implementations, at step/operation, the at least one processor retrieves/receives one or more user-defined parameters associated with the RNA polymerase of the target virus.

1912 At step/operation, the at least one processor performs a template loop (t-loop) analysis operation on the RNA polymerase. In some implementations, the at least one processor performs the t-loop analysis operation based at least in part on the retrieved/received one or more user-defined parameters. In some embodiments, the t-loop analysis operation comprises a sliding window operation as described in more detail herein. Example 6 below provides an example algorithm (Python script) for performing a t-loop analysis operation/sliding window operation in accordance with embodiments of the present disclosure.

1914 At step/operation, the at least one processor determines at least one optimal RNA sequence corresponding with the RNA polymerase of the target virus based on results (e.g., one or more determined values) from the t-loop analysis operation. In some embodiments, the at least one optimal RNA sequence comprises a stem-loop. In some examples, the stem-loop further comprises a stem portion with at least 5 base pairs (bps) in length and a loop portion with about 20 nucleotides in length, and wherein the loop portion matches a footprint of the RNA polymerase of the target virus. In some examples, the stem-loop is flanked by a 5′ promoter and a 3′ promoter RNA sequence of the target virus.

1916 At step/operation, the at least one processor outputs (e.g., via a display device) the at least one optimal RNA sequence. For example, the processor may output user-interface data to an end user via a graphical user interface or generate and provide (e.g., send, transmit) a text file. Additionally, and/or alternatively, the at least one processor can output at least one criterion of a composition (or instructions for engineering the composition) for administration to a subject based on the at least one optimal RNA sequence.

In one aspect, disclosed herein is a computer-implemented method for generating at least one optimal ribonucleic acid (RNA) sequence for reducing viral replication and/or inducing activation of an innate response to a target virus, the computer-implemented method comprising retrieving, by one or more processors, an RNA polymerase of the target virus, performing, by the one or more processors, a template loop (t-loop) analysis operation on the RNA polymerase, determining, by the one or more processors, at least one optimal RNA sequence corresponding with the RNA polymerase of the target virus based on results of the t-loop analysis operation, and outputting, by the one or more processors, a ΔG, a location of the ΔG, a t-loop structure, or combinations thereof. In some embodiments, the output comprises the at least one optimal RNA sequence.

In some embodiments, the computer-implemented method further comprises receiving, by the one or more processors, one or more user-defined parameters associated with the RNA polymerase of the target virus, and performing, by the one or more processors, the t-loop analysis operation based at least on the one or more user-defined parameters. In some embodiments, the t-loop analysis operation is used interchangeably with a sliding window operation. In some embodiments, the computer-implemented method further comprises determining, by the one or more processors, at least one criterion of a composition for administration to a subject based on the at least one optimal RNA sequence.

In some embodiments, the method can be used to optimize virus genome sequences and alter their growth kinetics. In some embodiments, the method can be used to optimize the growth of viral strains used for vaccine production.

A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

The influenza A virus (IAV) RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVG) and mini viral RNAs (mvRNA), during infection. Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Herein, evidence is provided that RNA structures, called template loops (t-loop), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are supportive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.

During an IAV infection, the virus introduces eight ribonucleoproteins (RNP) into the host cell nucleus. These RNPs consist of oligomeric viral nucleoprotein (NP), a copy of the viral RNA polymerase, and one of the eight segments of single stranded negative sense viral RNA (vRNA) that make up the viral genome. The vRNA segments range from 890 to 2341 nt in length, but all contain conserved 5′ triphosphorylated, partially complementary 5′ and 3′ termini. These termini serve as promoter for the RNA polymerase, but also as agonist of RIG-I. In the context of an RNP, the termini are bound by the RNA polymerase subunits PB1, PB2, and PA, and during viral replication, a second RNA polymerase is recruited to the RNP to encapsulate nascent RNA. It has been contemplated that binding of the viral RNA polymerase to the vRNA termini reduces RIG-I binding to the vRNA segments and it is not clear when or where RIG-I gains access to viral RNAs.

In addition to full-length vRNA and cRNA molecules, the viral RNA polymerase can produce aberrant RNAs that are shorter than the vRNA or cRNA template from which they derive. Such aberrant RNAs include defective viral genomes (DVGs) and mini viral RNAs (mvRNA), which contain internal deletions between the conserved 5′ and 3′ termini. Both DVGs and mvRNAs can bind RIG-I and activate innate immune responses, but only DVGs require viral NP during viral replication while mvRNAs do not. It is presently not fully understood what determines the ability of DVGs and mvRNAs to activate RIG-I or how they are made. Interestingly, the RNA polymerases of highly pathogenic avian H5N1 and pandemic 1918 H1N1 IAV produce higher mvRNA levels than the RNA polymerase of lab adapted H1N1 IAV, showing that there is a correlation between adaptive mutations in the RNA polymerase, mvRNA production, and innate immune activation in infections with highly pathogenic IAV.

1 FIG.B 1 FIG.B 6 FIG. Herein, this example examines the role of mvRNAs in innate immune activation in more detail. mvRNAs are generated, in part, via a copy-choice mechanism that results in the loss of an internal genome segment sequence, similar to what has been observed for DVGs (). As a result, RNA sequences or structures that do not normally reside side-by-side in the full-length genome segments are brought closer to each other in the nascent RNA, resulting in the formation of novel RNA structures (). Once generated, mvRNAs can be replicated by the viral RNA polymerase in the absence of NP. Inherently, the RNA polymerase is not impaired by RNA structures in an mvRNA template and it can replicate and transcribe an mvRNA containing a copy of the aptamer Spinach, a highly-structured RNA capable of stabilizing the fluorophore DFHBI (). However, it is not known if other RNA secondary structures or certain sequence combinations could impair mvRNA replication or play a role in the activation of the innate immune response during IAV infection, as for instance has been observed for paramyxovirus infections. Herein, a previous model is advanced on the effect of mvRNAs on the innate immune response and provide evidence that mvRNAs capable of inducing innate immune responses contain RNA structures that can reduce the activity of the IAV RNA polymerase.

Induction of IFN-β Promoter Activation by mvRNAs is Sequence Dependent

1 FIG.B mvRNAs bind RIG-I and activate the MAVS signaling cascade, but it is unclear what determines whether an IAV mvRNA is an inducer of the innate immune response. To systematically investigate if the sequence or secondary structure of an mvRNA can affect IAV RNA polymerase activity and innate immune activation, five segment 5-derived mvRNA templates were engineered. Each engineered mvRNA had a length of 71 nt (NP71.1-NP71.5), but a different internal sequence (Table 1). The positive control mvRNAs were 56- and 76-nt long mvRNAs, which were previously constructed from segment 5 (NP56 and NP76, respectively), while the negative control mvRNA was a 47-nt long mvRNA derived from segment 5 (NP47) that is unable to bind RIG-I and induce a strong IFN signal. To validate the test setup, increasing amounts of in vitro transcribed NP76 was transfected into HEK 293T cells and a strong increase in IFN-β promoter activity was found that saturated at a ˜50-fold induction, while the NP47 induced a lower activity (). These observations show how these RNAs differentially induce IFN-β promoter activity when they are transfected into the cytoplasm.

1 7 FIGS.C and 7 FIG. Subsequently the IFN-β promoter activation was validated by the NP47, NP56 and NP76 mvRNA templates during replication by the IAV RNA polymerase. To this end, plasmids expressing the viral RNA polymerase subunits PB1, PB2 and PA, a plasmid expressing NP, and a plasmid expressing the NP76 template mvRNA were transfected into HEK 293T cells. Primer extension analysis showed efficient amplification of NP47, NP56 and NP76, as well as the production of several smaller aberrant RNA products that were shorter than template the mvRNA in the case of NP56 and NP76 (). IFN-β promoter activation by NP56 and NP76, but not NP47 was observed. These results thus indicate that like the full-length vRNA segments, mvRNAs themselves can also serve as template for aberrant RNA synthesis. Fractionation of cells in which NP76 was replicated showed that the NP76 mvRNA template was present in the nuclear, cytoplasmic, and mitochondrial fractions, whereas the aberrant RNAs produced from the NP76 template were present in the nucleus only (). Since IAV RNA is predominantly detected in the cytoplasm of the host cell, these results show that the mvRNA template, and not aberrant products shorter than the template mvRNA, play a role in innate immune activation.

1 FIG.C 8 FIG.A 1 FIG.C 1 FIG.D 8 FIG.B Following the characterization of these assays, the replication and IFN-β promoter activation was analyzed by the engineered mvRNA templates and found that three of these templates were efficiently replicated (NP71.1, NP71.4 and NP71.5), while the other two (NP71.2 and NP71.3) were not (). Interestingly, among the engineered mvRNAs, templates that were poorly replicated showed higher IFN-β promoter activity and aberrant RNA synthesis (i.e., the production of RNA products containing deletions relative to the template;) than the three mvRNA templates that were efficiently replicated (). RT-qPCR analysis of cells replicating NP71.1 and NP71.2 confirmed that endogenous IFN-β mRNA levels were increased when NP71.2 was replicated (). To confirm that the NP71.2 had the ability to induce innate immune activation during viral infection, NP71.1 and NP71.2 was pre-expressed in absence of viral RNA polymerase and NP in HEK 293T cells. After 24 hours, the cells were infected with 3 MOI influenza virus A/WSN/1933 (H1N1) for 8 hours. As shown in, pre-expression of NP71.1 and NP71.2 had no effect on segment 6 replication or PB1 protein expression. In addition, phosphorylation of IRF3 after pre-expression of NP71.1 but not NP71.2 was observed. While this is shows MAVS signaling pathway activation through replication of mvRNA by the viral RNA polymerase, amplification of the exogenous mvRNAs could not be detected because they need to compete with the eight endogenous vRNA templates for binding to the viral RNA polymerase expressed by the virus. Thus, it was not determined whether the engineered mvRNAs affect the MAVS signaling pathway in the same way during viral infection as in the RNP reconstitution experiments.

Renilla 1 FIG.C 8 FIG.C To exclude that a differential recognition of the engineered mvRNAs by host pathogen receptors of the host cell was responsible for the observed increased IFN-β promoter activity on the NP71.2 and NP71.3 mvRNAs, total RNA was isolated from HEK 293T transfections and re-transfected equal amounts of these RNA extracts together with IFN-β andreporter plasmids into HEK 293T cells. Re-transfection of NP71.1-NP71.5 showed an inverse pattern of IFN-β promoter activation in comparison to, whereby abundant mvRNAs induced more IFN-β promoter activity than the least abundant mvRNAs (), showing that there is no inherent difference between the mvRNA in their ability to activate IFN-β promoter activity. Instead, these results indicated that impaired active viral replication determines whether an mvRNA will activate innate immune signaling in the context of an RNP.

−/− −/− −/− −/− 9 FIG.A 9 FIG.B 9 FIG.C 9 FIG.C To verify that the different replication efficiencies had not been the result of the effect of NP71.2 and NP71.3 on the innate immune response, these mvRNAs and the WSN RNA polymerase were also expressed in MAVSIFN::luc HEK 293 cells. These MAVScells do not express endogenous MAVS (), blocking any RIG-I mediated innate immune signaling, but overexpression of a MAVS-FLAG plasmid still triggers IFN-β promoter activity, indicating that the IFN-β reporter is still functional (). Expression of NP71.1-NP71.5 in the MAVScells did not induce IFN-β promoter activity (). Subsequent primer extension analysis showed that the differences in replication between NP71.1-NP71.5 had been maintained in the MAVScells (), demonstrating that the differential replication efficiency is not dependent on the innate immune response.

10 FIG. 10 FIG. To investigate whether the effect of the NP71.3 and NP71.4 mvRNAs was specific to the WSN polymerase, these mvRNAs were expressed alongside the pandemic H1N1 A/Brevig Mission/1/18 (abbreviated as BM18) or the highly pathogenic avian H5N1 A/duck/Fujian/01/02 (abbreviated as FJ02) RNA polymerases. The BM18 and FJ02 RNA polymerases were impaired on the NP71.2 and NP71.3 mvRNA templates and triggered a stronger IFN-β promoter activity on the NP71.3 template relative to the NP71.1 template (). The BM18 RNA polymerase also produced short aberrant RNA products, while the FJ02 RNA polymerase did not, despite inducing IFN-β promoter activity (). Together, these results show that the mvRNA template is the innate immune agonist, rather than the aberrant RNA products derived from the mvRNA template, and that innate immune activation is dependent on a sequence-specific interaction between the active IAV RNA polymerase and the mvRNA template.

2 FIG.A 2 FIG.A 2 FIG.A 11 11 FIGS.A andB 2 FIG.B 11 FIG.C 11 FIG.D 2 FIG.B The viral RNA template enters and leaves the active site of the IAV RNA polymerase as a single strand through the entry and exit channels, respectively (). However, the IAV genome contains various RNA structures that need to be unwound. Moreover, unwinding of these structures may lead to the formation of transient RNA structures upstream or downstream of the RNA polymerase that may modulate RNA polymerase activity (), while base pairing between a part of the template that is entering the RNA polymerase and a part of the template that has just been duplicated may trap the RNA polymerase in a template loop (t-loop) (). To systematically analyze what (transient) RNA structures are present during replication, a sliding-window algorithm was used to calculate the minimum free energy (ΔG) for every putative t-loop as well as every putative secondary RNA structure upstream and downstream of the RNA polymerase (). For each position analyzed, 20 nt were excluded from the folding analysis for the footprint of the IAV RNA polymerase and 12 nt from the 5′ terminus, which is stably bound by the RNA polymerase prior to replication termination. As shown inand, the analysis revealed that NP71.2 and NP71.3 are unique among the engineered mvRNA templates in forming t-loop structures around nucleotide 29 of the positive sense replicative intermediate (cRNA), but not the negative sense (), showing that t-loops in the positive sense mvRNA template modulate RNA polymerase activity. The likelihood that the t-loops form in the context of other secondary structures was calculated as the difference (ΔΔG) between the computed ΔG values for the individual structures ().

2 12 FIGS.B andA 2 FIG.B 2 12 FIGS.C andB 2 FIG.C 12 FIG.C 12 12 FIGS.C andD 1 FIG.C 6 FIG. 11 FIG.E To confirm that t-loops affect RNA polymerase processivity and IFN-β promoter activity, two A-U base pairs of the NP71.2 t-loop duplex were replaced with two G-U base pairs, creating NP71.6 (). Using the sliding window analysis, this mutation was confirmed to make t-loop formation near nucleotide 29 less favorable (). Following expression of NP71.1, NP71.2 and NP71.6, replication of the NP71.6 mvRNA was increased relative to the NP71.2 mvRNA, our control mvRNAs, and the NP71.1 mvRNA (). In addition, destabilization of the t-loop reduced the induction of the IFN-β promoter activity (). By contrast, mutating the stem of the t-loop of the NP71.2 mvRNA template such that the t-loop around nucleotide 29 was maintained (; NP71.7-8), replication remained reduced and IFN-β reporter activity increased relative to the NP71.1 mvRNA (). Replication of mvRNAs with a t-loop led to the production of short aberrant RNA products that likely contained internal deletions. However, increases in aberrant RNA levels were not correlated with increases in IFN-β reporter activity, in line with the results inand, and indicated that the mvRNA template is the agonist of IFN-β reporter activity. Analysis of control mvRNA templates showed that NP56 and NP76 contain weak t-loops in the first half of both the positive and negative sense template, while stronger t-loops exist in the second half for the NP56 template (). Together these results indicate that t-loops can negatively affect IAV RNA synthesis and stimulate innate immune signaling during IAV replication.

3 FIG.A 3 FIG.B 1 2 FIGS.C andC 3 FIG.B The mvRNAs NP71.2 and NP71.3 contain a t-loop in the first half of the positive sense mvRNA template. To confirm that t-loops also affect RNA polymerase activity in the negative sense, three additional 71-nt long mvRNA templates were engineered with t-loops in different locations of the template (NP71.10-12) (Table 1;). Expression of these mvRNA templates together with the subunits of the viral RNA polymerase in HEK 293T cells led to strongly reduced NP71.10 and NP71.11 mvRNA levels and slightly reduced NP71.12 mvRNA levels (). In line with other results (), IFN-β promoter activity was increased for the NP71.11 and NP71.12 templates relative to the NP71.1 mvRNA, while the NP71.10 mvRNA did not induce IFN-β promoter activity because it was too poorly or not fully replicated ().

3 FIG.C 3 FIG.A 3 FIG.B To investigate if t-loops affect RNA polymerase processivity in vitro, the WSN RNA polymerase was purified from HEK 293T cells using tandem-affinity purification (TAP) and incubated the enzyme with the NP71.1 and NP71.10 mvRNA templates in the presence of NTPs. Following denaturing PAGE and autoradiography, a main product of approximately 71 nt in reactions was observed containing the NP71.1 control mvRNA (). By contrast, incubations with the NP71.10 mvRNA template resulted in products up to approximately 27 nt in length, in agreement with the location of the t-loop in the first half of the mvRNA template (). Moreover, the observed partial extension of the product offered a possible explanation for the reduced RNA levels in cell culture and the lack of IFN-β promoter activity induction by the NP71.10 template ().

13 FIG.A 13 FIG.A To investigate if t-loop containing templates remained stably bound to the RNA polymerase or triggered template dissociation, mOrange-tagged RNA polymerase was immobilized on magnetic RFP-trap beads, and incubated these immobilized complexes with radiolabeled template. After removal of unbound template by three washes with binding buffer, ApG and nucleotides were added to initiate RNA synthesis and complexes incubated at 30° C. for 15 min. Next, the immobilized complexes were washed three times to remove dissociated RNA, and the reactions stopped with formaldehyde/EDTA loading dye. Analysis of the bound and unbound radiolabeled RNA levels by dot blot and autoradiography showed no difference between the NP71.1, NP71.10 and NP71.11 templates (). To rule out that released template were rebound upon dissociation from the RNA polymerase, excess unlabeled NP71.1 template was added as RNA polymerase trap at the start of the reaction. Again, no difference between the templates was observed ().

13 FIG.B To confirm that the immobilized RNA polymerases were active, RNA polymerase bound to unlabeled template mvRNA was immobilized on magnetic beads as described above. Next, ApG, NTPs, and radiolabeled GTP was added and incubated the immobilized complexes at 30° C. for 15 min. Following removal of unincorporated NTPs by three washes with binding buffer, the nascent RNA in solution as well as associated with the immobilized complexes was analyzed by denaturing PAGE and autoradiography. As shown in, partially extended and full-length nascent RNAs remained associated with the immobilized RNA polymerases. Partially extended nascent RNAs were also found in the unbound fraction. Addition of inactive RNA polymerase (PB1a) to serve as encapsulating polymerase in RNA polymerase dimers increased the release of partially extended nascent RNAs, but not the release of full-length RNAs. Together, these results show that t-loops do not induce template release upon RNA polymerase stalling and that partially extended nascent strands can be separated by RNA polymerase from the template strand and released.

PB1 K669A Increases t-Loop Sensitivity and IFN-β Promoter Activation

2 4 FIGS.A andA 2 4 FIGS.A andA 2 4 FIGS.A andA 4 FIG.A In the IAV RNA polymerase elongation complex, the 3′ terminus of the template is guided out of the template exit channel via an exit groove on the outside of the thumb subdomain. This groove consists of PB1 and PB2 residues, and leads to promoter binding site B (). Since this exit groove and the template entry channel reside next to each other at the top of the RNA polymerase (), perturbation of the path of the 3′ terminus out of the exit channel may stabilize t-loop formation, reduce RNA polymerase activity, and increase IFN-β promoter activation (). It was observed that avian adaptive mutations of highly pathogenic IAV RNA polymerases that increase IFN promoter activation in vitro, such as PB2 M81T (), reside next to the template exit groove. It was therefore contemplated that other mutations near the template exit channel may make the IAV RNA polymerase more sensitive to t-loops.

3 FIG.B 3 FIG.C 3 3 FIGS.D andE 3 3 FIGS.D andE 3 3 FIGS.B andE To test if dysregulation of the exit groove leads to more IFN-β promoter activation, PB1 lysine 669, which resides at the start of the exit groove (), were mutated to alanine (K669A). Mutation of this residue had no effect on RNA polymerase activity in the presence of a full-length segment 6 template () or the NP71.1 and NP71.6 mvRNA templates that do not contain a stable t-loop (). However, when the K669A mutant was expressed together with the NP71.2, NP71.11 or NP71.12 mvRNAs, which do contain a t-loop in either the positive or negative sense, the K669A mutant displayed greatly reduced RNA polymerase activity (), showing that the K669A mutation increases the processivity defect induced by t-loops. In contrast, the effect of K669A on IFN-β promoter activity was more difficult to interpret, because while the IFN-β promoter activity was considerably increased on the t-loop containing templates (), the K669A mutant also induced significantly higher IFN-β promoter activity relative to the wild-type RNA polymerase on the control templates. These results show that the K669A mutation has two effects: increase the base-level of the RNA polymerase to trigger FN-β promoter activity on templates without a known or destabilized t-loop through an unknown mechanism, and make the RNA polymerase more sensitive to disruption by a t-loop and trigger additional IFN-β promoter activity through this mechanism.

Differential IFN-β Promoter Activation by Natural mvRNAs

5 5 FIGS.A andB mvRNAs are produced during IAV infection in vitro and in vivo. To study how their sequence and abundance varies, RNA extracted from ferret lungs was examined 1 day post infection with BM18 for 1 day and A549 cells infected with WSN for 8 hours (see Examples 2-5 for mvRNA sequences). Although no quantitative comparisons can be made due to the different infection conditions, a strikingly similar variation in mvRNA sequence and abundance was found ().

14 FIG.A 5 FIG.C 5 FIG.D 14 FIG.B 5 FIG.D 1 FIG. To investigate the implications of these mvRNA differences on the activation of the IFN-β promoter, ten WSN segment 2 mvRNAs (randomly selected over a range of copy numbers and lengths;) were cloned into pPolI plasmids (mvRNAs A-J; Table 2). Analysis of the ΔΔG values for these mvRNAs revealed potential t-loops in the first half of the sequence for mvRNAs C, D, H and J, and t-loops in the second half of the sequence for mvRNAs E, F and G (). Subsequent expression of the authentic WSN mvRNAs alongside the WSN RNA polymerase in HEK 293T cells showed significant differences in mvRNA amplification (). These differences were correlated with the abundance detected by next generation sequencing (NGS) for seven of the cloned mvRNAs (). In addition, replication of mvRNAs C, D and J leads to the appearance of products shorter than the template mvRNA (), and the appearance of these products is correlated with a reduced replication of the template mvRNA, in line with the findings in.

5 FIG.D 1 FIG.C 5 FIG.E 1 FIG. 5 FIG.F 15 15 FIGS.A andB To investigate if the different segment 2 mvRNA levels influenced the innate immune response, the IFN-β promoter activity was measured. IFN-β promoter activity varied greatly, with mvRNAs C, D and J inducing the strongest response (). Templates I and G, the two shortest mvRNAs at 52 and 40 nt long, induced the lowest IFN-β promoter activity, in line with previous observations that short mvRNAs<56 nt do not stimulate RIG-I and. With mvRNAs I and G excluded due to their short size, these observations indicate that the IFN-β promoter activity is negatively correlated with mvRNA template level for mvRNAs>56 nt (). Moreover in, t-loops in the first half of the template affect RNA polymerase processivity, the IFN-β promoter activity was negatively correlated with the mean ΔΔG of the first half of the template (). Weaker correlations were observed between the mvRNA length and IFN-β induction, or the mvRNA length and mvRNA replication ().

16 FIG.A 16 FIG.B 16 FIG.C −/− To exclude that a differential recognition of the mvRNAs was responsible for the observed anti-correlation, total RNA was isolated from HEK 293T transfections and re-transfected equal amounts of these RNA extracts into HEK 293T cells. As shown in, no significant difference among the segment 2 mvRNAs longer than 56 nt was observed. The mvRNAs G and I failed to induce a strong response due to their short length. To exclude that the different mvRNA levels had been the result of their different effects on the innate immune response, the segment 2 mvRNAs were also expressed in MAVSIFN::luc HEK 293 cells. Following expression of the segment 2 mvRNAs, no IFN-β promoter activity was observed (). Primer extension analysis showed no significant reduction in mvRNA steady-state levels compared to wildtype cells (), indicating that the replication of authentic mvRNAs is not impacted by innate immune activation.

17 FIG. To confirm that mvRNAs from other viral segments have differential effects on the innate immune response, two segment 3 mvRNAs and four segment 4 mvRNAs (Table 3) were cloned from the mvRNA sequences identified during infection into pPol expression plasmids and transfected these plasmids into HEK 293T cells. As shown in, PA and HA mvRNAs induced both high and low levels of IFN-β promoter activity compared our NP71.1 control. Together, these results indicate that viral infections produce mvRNAs with different mechanisms to induce IFN-β promoter activity, and that t-loops play a key role in the mvRNAs inducing IFN-β promoter activity by affecting the ability of the RNA polymerase to efficiently replicate them.

2 FIG.A 3 FIG.C Two factors important for inducing an innate immune response in IAV infections are active viral replication and the binding of viral RNA molecules to RIG-I. Herein, the effect of IAV mvRNAs, which do not need viral NP to be replicated by the viral RNA polymerase, was examined. Evidence is provided that impeded viral RNA polymerase processivity by t-loops is a mechanism that contributes to the activation of innate immune signaling by mvRNAs. While there is no direct assay to measure or visualize t-loop formation in mvRNAs yet and can thus not rule out other or additional mechanisms, it is postulated that t-loops form when the 3′ terminus or a sequence near the 3′ terminus of the template can hybridize with an upstream part of the template (). RNA polymerase is thought to unwind a single t-loop, but the formation of several successive t-loops in the first half of the mvRNA stalls the RNA polymerase (). It is presently still unclear why a strong correlation between reduced processivity and t-loops is observed with t-loops in the first half of the template and not with downstream t-loops.

4 FIG.G 3 FIG.D 7 FIG. It is unclear how RIG-I gains access to the t-loop containing mvRNA once the polymerase has stalled (). RNA polymerase stalling does not result in release of the RNA template from the active site () because the RNA polymerase remains associated with the 5′ terminus of the template prior to replication termination. This means that it is still unclear how mvRNA templates accumulate in the cytoplasm and mitochondria ().

4 FIG. One mechanism is that t-loops affect influenza RNA polymerase activity on full-length viral RNAs or DVGs. However, it is more likely that t-loops form only on partially formed RNPs or NP-less templates, since NP may modulate the presence and location of secondary RNA structures. During viral RNA synthesis, NP dissociates and binds viral RNA in a manner that is coordinated by the viral RNA polymerase. When NP levels are reduced, aberrant RNPs or NP-less RNA products form in which secondary RNA structures that are absent in the presence of NP contribute to t-loop formation and RNA polymerase stalling. Indeed, this model explains how reduced viral NP levels stimulate aberrant RNA synthesis and innate immune activation. A mutation near the template exit channel increases the RNA polymerase sensitivity to t-loops (). It was previously observed that avian adaptive mutations, such as PB2 N9D or M81T, reside near the template exit channel of highly pathogenic IAV RNA polymerases and that they stimulate IFN-β promoter activity. Thus, mutations make the RNA polymerase more sensitive to mvRNAs, which are produced at high levels by highly pathogenic IAV RNA polymerases, and this sensitivity leads to increased RNA polymerase stalling by t-loops and IFN-β promoter activation.

During viral infection, mvRNA molecules of various lengths and abundances are produced. It was found that mvRNA abundance is not the best estimate for innate immune activation. An updated model in which mvRNAs that are poorly replicated contribute most to the activation of the innate immune system is contemplated, and thus that activation of the innate immune response is dependent on a template sequence context.

pcDNA3-based plasmids expressing influenza A/WSN/33 (H1N1) proteins PB1, PB2 PA, NP, PB2-TAP and the active site mutant PB1a (D445/D446A). Mutation K669A was introduced into the pcDNA3-PB1 plasmid by site-directed mutagenesis. mvRNA templates were expressed under the control of the cellular RNA polymerase I promoter from pPolI plasmids. PB1 mvRNA templates were generated by site-directed mutagenesis PCR deletion of pPolI-PB1. Short vRNA templates were created based on the pPolI-NP47 plasmid using the SpeI restriction site.

Renilla Renilla Firefly luciferase reporter plasmid under the control of the IFNB promoter (pIFΔ(-116)lucter) and constitutively expressingluciferase plasmid (pcDNA3-). The MAVS-FLAG expression vector and corresponding empty vector were cloned based on the pFS420, using the MAVS WT plasmid (pEF-HA-MAVS).

Mycoplasma −/− Human embryonic kidney (HEK) 293T, Madin-Darby Canine Kidney (MDCK), and A549 cells were originally sourced from the American Type Culture Collection. All cells were routinely screened for. HEK293 wild-type and MAVScells expressing luciferase under the control of the IFNB promoter were a kind gift from Dr J. Rehwinkel. All cell cultures were grown in Dulbecco's Modified Eagle Medium (DMEM) (Sigma) with 10% fetal bovine serum (FBS) (Sigma) and 1% L-Glutamine (Sigma). Transfections of HEK293T or HEK293 cell suspensions were performed using Lipofectamine 2000 (Invitrogen) and Opti-Mem (Invitrogen) following the manufacturer's instructions, and transfection of confluent, adherent HEK 293T cells were performed using PEI (Sigma) and Opti-Mem. Infections were performed at MOI 3.

IAV proteins were detected using rabbit polyclonal antibodies anti-PB1 (GTX125923; GeneTex), anti-PB2 (GTX125926; GeneTex), and anti-NP (GTX125989; GeneTex) diluted 1:1000 in TBS™ (TBS/0.1% Tween-20 (Sigma)/5% milk). Cellular proteins were detected using the rabbit polyclonal antibodies anti-GAPDH (GTX100118; GeneTex) diluted 1:4000 in TBS™, and anti-RNA Pol II (ab5131; Abcam) diluted 1:100 in TBS™; the mouse monoclonal antibodies anti-MAVS E-3 (sc-166583; Santa Cruz) diluted 1:200 in TMS™, and Mito tracker [113-1](ab92824; Abcam) diluted 1:1000 TBS™; and the rat monoclonal antibody anti-tubulin (MCA77G; Bio-Rad) diluted 1:1000 in TBS™. Mouse monoclonal antibody anti-FLAG M2 (F3165; Sigma) diluted at 1:2000 was used to detect MAVS-FLAG. Secondary antibodies IRDye 800 Donkey anti-rabbit (926-32213; Li-cor), IRDye 800 Goat anti-mouse (926-32210; Li-cor), IRDye 680 Goat anti-mouse (926-68020; Li-cor), and IRDye 680 Goat anti-rat (926-68076; Li-cor), were used to detect western signals with a Li-cor Odyssey scanner.

11 11 FIGS.A andB Infections and RNA analyses using primer extensions were performed as described previously. mvRNA identification from next generation sequencing data was essentially performed, using data deposited in the Sequence Read Archive under accession number SUB3758924. Aberrant RNA products observed in various experiments were gel extracted, Topo cloned and sequenced using Sanger sequencing. Alignments were analyzed using Clustal Omega and visualized using Espript 3. T-loop analysis was performed using a custom Python script. Briefly, 20 nt of the template sequence were blocked off to represent the footprint of the viral RNA polymerase. This footprint was then moved in 1 nt increments along the template (). T-loop formation was assessed by computing the ΔG of duplex formation between a stretch of 10 nt upstream of the footprint and 10 nt downstream of the footprint. The formation of upstream and downstream structures was computed for 24 nt windows (the footprint of NP) upstream and downstream of the moving footprint. The ΔΔG was computed by subtracting The ViennaRNA package commands duplex-fold and cofold were used to compute the ΔG values.

Renilla Renilla To measure IFN expression in RNP reconstituted HEK293T or HEK293 cells, luciferase assays were carried out 24 h post-transfection. RNP reconstitutions were carried out in a 24-well format by transfecting 0.25 μg of the plasmids pcDNA3-PB1, pcDNA3-PB2, pcDNA3-PA, pcDNA3-NP and a pPolI plasmid expressing a mvRNA template. HEK293T and HEK293 cells were additionally co-transfected with 100 ng of the plasmids pIFΔ(-116)lucter and pcDNA3-. Cells were harvested in PBS and resuspended in an equal volume of Dual-Glo Reagent (Promega) followed by Dual-Glo Stop & Glo reagent (Promega). Firefly andluminescence were measured after 10 minutes incubation with each reagent respectively as per manufacturer's instructions for the Dual-Glo Luciferase Assay System (E2920, Promega) using the Glomax luminometer (Promega).

Influenza virus A/WSN/33 (H1N1) recombinant polymerases were purified from HEK293T cells. Ten cm plates of adherent cells were transfected with 3 μg of pcDNA3-PB1, pcDNA3-PB2-TAP and pcDNA3-PA with PEI (Sigma). Forty-eight hours post-transfection, cells were harvested in PBS and lysed on ice for 10 min in 500 μl lysis buffer (50 mM hepes pH 8.0, 200 mM NaCl, 25% glycerol (Sigma), 0.5% Igepal CA-630 (Sigma), 1 mM β-mercaptoethanol (Bio-Rad), 1×PMSF (Sigma), 1× Protease Inhibitor cocktail tablet (Roche). Lysates were cleared by centrifugation at 17000 g for 5 min at 4° C., diluted in 2 ml NaCl (Sigma), and bound to pre-washed IgG Sepharose beads (Cytiva) for 2 h at 4° C. Beads were pre-washed 3× in binding buffer (10 mM Hepes pH 8.0, 0.15 M NaCl, 0.1% Igepal CA-630, 10% glycerol, 1×PMSF). After binding, beads were washed 3× in binding buffer and 1× in cleavage buffer (10 mM Hepes pH 8.0 (Sigma), 0.15M NaCl, 0.1% Igepal CA-630, 10% glycerol, 1×PMSF, 1 mM DTT). Beads were cleaved with AcTEV protease (Invitrogen) overnight at 4° C., and cleared by centrifugation at 1000 g for 1 min. Activity assays using immobilized RNA polymerase were performed using an RNA polymerase with an mOrange-tag on the PB2 subunit. The purified polymerase was immobilized using magnetic RFP-trap beads (Chromotek).

Fractionation of transfected cells into cytoplasmic, mitochondrial, and nuclear components was carried out using the Abcam Cell Fractionation Kit (Abcam) following the manufacturer's instructions, with volumes adjusted based on the number of cells. Samples of unfractionated whole cells in Buffer A were retained as input controls. Whole cells and sub-cellular fractions were dissolved in Trizol, for RNA extraction and analyzed as described above, or in 10%-SDS protein-loading buffer, for protein expression analysis by SDS-PAGE and western blot.

−/− Statistical testing was carried out using GraphPad Prism 9 software. Error bars represent standard deviations, and either individual data or group mean values are plotted. One-way analysis of variance (ANOVA) with Dunnett's test for multiple comparisons was used to compare multiple-group means to a normalized mean (e.g., IFN induction or RNA template replication). Two-way ANOVA with Sidak's test for multiple comparisons was used to compare multiple pairs of group means (e.g., between two cell types, HEK293 WT to HEK293 MAVS).

1) -WSN_NP_61 SEQ ID NO: 31 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATAGAAAAATACCCT TGTTTCTACT 2) -WSN_PB1_57 SEQ ID NO: 32 AGCGAAAGCAGGCAAACCATTTGAATGGATTTCATGAAAAAATGCCTTGT TTCTACT 3) -WSN_PB1_57 SEQ ID NO: 33 AGCGAAAGCAGGCAAACCATTTGAATGTCCTTCATGAAAAAATGCCTTGT TTCTACT 4) -WSN_PB1_66 SEQ ID NO: 34 AGCGAAAGCAGGCAAACCATTTGAATGTTTAGCTTGTCCTTCATGAAAAA ATGCCTTGTTTCTACT 5) -WSN_NP_65 SEQ ID NO: 35 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCTAAAGAAAAAT ACCCTTGTTTCTACT 6) -WSN_HA_44 SEQ ID NO: 36 AGCAAAAGCAGGGGAATATAAGGAAAAACACCCTTGTTTCTACT 7) -WSN_NA_70 SEQ ID NO: 37 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCTGACAAGTAGTTTGTTCA AAAAACTCCTTGTTTCTACT 8) -WSN_PB1_62 SEQ ID NO: 38 AGCGAAAGCAGGCAAACCATTTGAATAGCTTGTCCTTCATGAAAAAATGC CTTGTTTCTACT 9) -WSN_NP_70 SEQ ID NO: 39 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATTAAAGA AAAATACCCTTGTTTCTACT 10) -WSN_PB1_64 SEQ ID NO: 40 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTAAAAAAT GCCTTGTTTCTACT 11) -WSN_PB1_69 SEQ ID NO: 41 AGCGAAAGCAGGCAAACCATTIGAATGGATGTTAGCTTGTCCTTCATGAA AAAATGCCTTGTTTCTACT 12) -WSN_NA_69 SEQ ID NO: 42 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 13) -WSN_PB1_62 SEQ ID NO: 43 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTAAAATGC CTTGTTTCTACT 14) -WSN_PB1_68 SEQ ID NO: 44 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTCATGAAA AAATGCCTTGTTTCTACT 15) -WSN_HA_60 SEQ ID NO: 45 AGCAAAAGCAGGGGAAATTAGGATTTCAGAAATATAAGGAAAAACACCCT TGTTTCTACT 16) -WSN_PB1_68 SEQ ID NO: 46 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCTCCTTCATGAAA AAATGCCTTGTTTCTACT 17) -WSN_PB1_49 SEQ ID NO: 47 AGCGAAAGCAGGCAAACCATTTGAATTGAAAAAATGCCTTGTTTCTACT 18) -WSN_PB1_56 SEQ ID NO: 48 AGCGAAAGCAGGCAAACTTTAGCTTGTCCTTCATGAAAAAATGCCTTGTT TCTACT 19) -WSN_PB2_60 SEQ ID NO: 49 AGCGAAAGCAGGTCAATTATATTCAATATCGAATAGTTTAAAAACGACCT TGTTTCTACT 20) -WSN_PB1_60 SEQ ID NO: 50 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATATGAAAAAATGCCT TGTTTCTACT 21) -WSN_NP 74 SEQ ID NO: 51 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCTACGACAATTA AAGAAAAATACCCTTGTTTCTACT 22) -WSN_NA_73 SEQ ID NO: 52 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAACAAGTAGTTTGT TCAAAAAACTCCTTGTTTCTACT 23) -WSN_NA_62 SEQ ID NO: 53 AGCAAAAGCAGGAGTTTAAATGAATCCAAACTAGTTTGTTCAAAAAACTC CTTGTTTCTACT 24) -WSN_NA_63 SEQ ID NO: 54 AGCAAAAGCAGGAGTTTAACACCATTGACAAGTAGTTTGTTCAAAAAACT CCTTGTTTCTACT 25) -WSN_PB2_55 SEQ ID NO: 55 AGCGAAAGCAGGTCAATTATATTCCGAATAGTTTAAAAACGACCTTGTTT CTACT 26) -WSN_PB1_44 SEQ ID NO: 56 AGCGAAAGCAGGCAAACCATATGAAAAAATGCCTTGTTTCTACT 27) -WSN_PA_64 SEQ ID NO: 57 AGCGAAAGCAGGTACTGATTCAAATTGCTATCCATACTGTCCAAAAAAGT ACCTTGTTTCTACT 28) -WSN_NP_70 SEQ ID NO: 58 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATAGA AAAATACCCTTGTTTCTACT 29) -WSN_HA_46 SEQ ID NO: 59 AGCAAAAGCAGGGGAAAATAAAAAGAAAAACACCCTTGTTTCTACT 30) -WSN_HA_61 SEQ ID NO: 60 AGCAAAAGCAGGGGAAAATAAAGATTTCAGAAATATAAGGAAAAACACCC TTGTTTCTACT 31) -WSN_PB1_65 SEQ ID NO: 61 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTGAAAAAA TGCCTTGTTTCTACT 32) -WSN_PB1_61 SEQ ID NO: 62 AGCGAAAGCAGGCAAACCATTTGAATGGTTGTCCTTCATGAAAAAATGCC TTGTTTCTACT 33) -WSN_PB1_67 SEQ ID NO: 63 AGCGAAAGCAGGCAAACCATTTGAATGATTTAGCTTGTCCTTCATGAAAA AATGCCTTGTTTCTACT 34) -WSN_PB1_60 SEQ ID NO: 64 AGCGAAAGCAGGCAAACCATTTGTAGCTTGTCCTTCATGAAAAAATGCCT TGTTTCTACT 35) -WSN_PB1_64 SEQ ID NO: 65 AGCGAAAGCAGGCAAACCATGTGAATTTAGCTTGTCCTTCATGAAAAAAT GCCTTGTTTCTACT 36) -WSN_PA_66 SEQ ID NO: 66 AGCGAAAGCAGGTACTGATTTACTATTTGCTATCCATACTGTCCAAAAAA GTACCTTGTTTCTACT 37) -WSN_NP 69 SEQ ID NO: 67 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC AAATACCCTTGTTTCTACT 38) -WSN_NP_70 SEQ ID NO: 68 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC AAAATACCCTTGTTTCTACT 39) -WSN_NP_66 SEQ ID NO: 69 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCTTAAAGAAAAA TACCCTTGTTTCTACT 40) -WSN_NP_64 SEQ ID NO: 70 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATTAAAGAAAAATA CCCTTGTTTCTACT 41) -WSN_NP_57 SEQ ID NO: 71 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACAAAAATACCCTTGT TTCTACT 42) -WSN_NP 59 SEQ ID NO: 72 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTTAAAGAAAAATACCCTT GTTTCTACT 43) -WSN_NP_55 SEQ ID NO: 73 AGCAAAAGCAGGGTAGATAATCACTCACAGAGAGAAAAATACCCTTGTTT CTACT 44) -WSN_NA_64 SEQ ID NO: 74 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCATAGTTTGTTCAAAAAAC TCCTTGTTTCTACT 45) -WSN_NA_71 SEQ ID NO: 75 AGCAAAAGCAGGAGTTTAAATGAATCCAACCATTGACAAGTAGTTTGTTC AAAAAACTCCTTGTTTCTACT 46) -WSN_HA_64 SEQ ID NO: 76 AGCAAAAGCAGGGGAATGAGATTAGGATTTCAGAAATATAAGGAAAAACA CCCTTGTTTCTACT 47) -WSN_PB2_62 SEQ ID NO: 77 AGCGAAAGCAGGTCAATTATATTCAATATGGAGAATAGTTTAAAAACGAC CTTGTTTCTACT 48) -WSN_PB2_57 SEQ ID NO: 78 AGCGAAAGCAGGTCAATTATATTCAATATGTAGTTTAAAAACGACCTTGT TTCTACT 49) -WSN_PB2_48 SEQ ID NO: 79 AGCGAAAGCAGGTCAATTATATTCATTAAAAACGACCTTGTTTCTACT 50) -WSN_PB2_54 SEQ ID NO: 80 AGCGAAAGCAGGTCAATTATAGTCGAATAGTTTAAAAACGACCTTGTTTC TACT 51) -WSN_PB2_49 SEQ ID NO: 81 AGCGAAAGCAGGTCAATTAGAATAGTTTAAAAACGACCTTGTTTCTACT 52) -WSN_PB2_58 SEQ ID NO: 82 AGCGAAAGCAGGTCAATCAATTAGTGTCGAATAGTTTAAAAACGACCTTG TTTCTACT 53) -WSN_PB1_68 SEQ ID NO: 83 AGCGAAAGCAGGCAAACCATTIGAATGGATGTCAATCCGACTTTACGAAA AAATGCCTTGTTTCTACT 54) -WSN_PB1_73 SEQ ID NO: 84 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTCCTTCA TGAAAAAATGCCTTGTTTCTACT 55) -WSN_PB1_61 SEQ ID NO: 85 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTAAATGCC TTGTTTCTACT 56) -WSN_PB1_63 SEQ ID NO: 86 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTAAAAATG CCTTGTTTCTACT 57) -WSN_PB1_80 SEQ ID NO: 87 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGAATTTAGCTTG TCCTTCATGAAAAAATGCCTTGTTTCTACT 58) -WSN_PB1_47 SEQ ID NO: 88 AGCGAAAGCAGGCAAACCATTTGAATGAAAAATGCCTTGTTTCTACT 59) -WSN_PB1_48 SEQ ID NO: 89 AGCGAAAGCAGGCAAACCATTTGAATGAAAAAATGCCTTGTTTCTACT 60) -WSN_PB1_64 SEQ ID NO: 90 AGCGAAAGCAGGCAAACCATTTGAATGTAGCTTGTCCTTCATGAAAAAAT GCCTTGTTTCTACT 61) -WSN_PB1_62 SEQ ID NO: 91 AGCGAAAGCAGGCAAACCATTTGATTAGCTTGTCCTTCATGAAAAAATGC CTTGTTTCTACT 62) -WSN_PB1_43 SEQ ID NO: 92 AGCGAAAGCAGGCAAACCATTTAAAAAATGCCTTGTTTCTACT 63) -WSN_PB1_40 SEQ ID NO: 93 AGCGAAAGCAGGCAAACTGAAAAAATGCCTTGTTTCTACT 64) -WSN_PB1_50 SEQ ID NO: 94 AGCGAAAGCAGGCAAACTTGTCCTTCATGAAAAAATGCCTTGTTTCTACT 65) -WSN_NS 47 SEQ ID NO: 95 AGCAAAAGCAGGGTGACAAAGAAATAAAAAACACCCTTGTTTCTACT 66) -WSN_NP_72 SEQ ID NO: 96 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGA GAAAAATACCCTTGTTTCTACT 67) -WSN_NP_88 SEQ ID NO: 97 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGG GAGTACGACAATTAAAGAAAAATACCCTTGTTTCTACT 68) -WSN_NP_91 SEQ ID NO: 98 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATAATGCA GAGGAGTACGACAATTAAAGAAAAATACCCTTGTTTCTACT 69) -WSN_NP 81 SEQ ID NO: 99 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAAGAGTACG ACAATTAAAGAAAAATACCCTTGTTTCTACT 70) -WSN_NP 62 SEQ ID NO: 100 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGGAAAAATACC CTTGTTTCTACT 71) -WSN_NP_57 SEQ ID NO: 101 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCAATACCCTTGT TTCTACT 72) -WSN_NP_58 SEQ ID NO: 102 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCAAATACCCTTG TTTCTACT 73) -WSN_NP_81 SEQ ID NO: 103 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGAATGCAGAGGAGTACG ACAATTAAAGAAAAATACCCTTGTTTCTACT 74) -WSN_NP_62 SEQ ID NO: 104 AGCAAAAGCAGGGTAGATAATCACTCATACGACAATTAAAGAAAAATACC CTTGTTTCTACT 75) -WSN_NP_49 SEQ ID NO: 105 AGCAAAAGCAGGGTAGAGACAATTAAAGAAAAATACCCTTGTTTCTACT 76) -WSN_NA_72 SEQ ID NO: 106 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAAAAGTAGTTTGTT CAAAAAACTCCTTGTTTCTACT 77) -WSN_NA__70 SEQ ID NO: 107 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGACAAGTAGTTTGTTCA AAAAACTCCTTGTTTCTACT 78) -WSN_NA_69 SEQ ID NO″ 108 AGCAAAAGCAGGAGTTTAAATGAATCCAAATTGACAAGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 79) -WSN_NA_45 SEQ ID NO: 109 AGCAAAAGCAGGAGTTTAAATTTCAAAAAACTCCTTGTTTCTACT 80) -WSN_NA_52 SEQ ID NO: 110 AGCAAAAGCAGGAGTTTAAATTAGTTTGTTCAAAAAACTCCTTGTTTCTA CT 81) -WSN_NA_62 SEQ ID NO: 111 AGCAAAAGCAGGAGTTTAAACCATTGACAAGTAGTTTGTTCAAAAAACTC CTTGTTTCTACT 82) -WSN_M_70 SEQ ID NO: 112 AGCGAAAGCAGGTAGATATTGAAAGATGAGTCTTCTAACCGAGGTCGTAA AAAACTACCTTGTTTCTACT 83) -WSN_M_47 SEQ ID NO: 113 AGCGAAAGCAGGTAGATATTGAAAGATAAAACTACCTTGTTTCTACT 84) -WSN_HA_52 SEQ ID NO: 114 AGCAAAAGCAGGGGAAAATAAAAACAACCAGAAAAACACCCTTGTTTCTA CT 85) -WSN_HA_49 SEQ ID NO: 115 AGCAAAAGCAGGGGAAAATAAAAACAAGAAAAACACCCTTGTTTCTACT 86) -WSN_HA_48 SEQ ID NO: 116 AGCAAAAGCAGGGGAAAATAAAAACAGAAAAACACCCTTGTTTCTACT 87) -WSN_HA_63 SEQ ID NO: 117 AGCAAAAGCAGGGGAAAATAAAAACATTTCAGAAATATAAGGAAAAACAC CCTTGTTTCTACT 88) -WSN_HA_67 SEQ ID NO: 118 AGCAAAAGCAGGGGAAAATAAAGATTAGGATTTCAGAAATATAAGGAAAA ACACCCTTGTTTCTACT 89) -WSN_HA_61 SEQ ID NO: 119 AGCAAAAGCAGGGGAAAATTAGGATTTCAGAAATATAAGGAAAAACACCC TTGTTTCTACT 90) -WSN_HA_58 SEQ ID NO: 120 AGCAAAAGCAGGGGAATAGGATTTCAGAAATATAAGGAAAAACACCCTTG TTTCTACT 91) -WSN_PB2_57 SEQ ID NO: 121 AGCGAAAGCAGGTCAATTATATTCAATATGGAAAGAAAAAACGACCTTGT TTCTACT 92) -WSN_PB2_69 SEQ ID NO: 122 AGCGAAAGCAGGTCAATTATATTCAATATGGAAAGAGTCGAATAGTTTAA AAACGACCTTGTTTCTACT 93) -WSN_PB2_67 SEQ ID NO: 123 AGCGAAAGCAGGTCAATTATATTCAATATGTAGTGTCGAATAGTTTAAAA ACGACCTTGTTTCTACT 94) -WSN_PB2_51 SEQ ID NO: 124 AGCGAAAGCAGGTCAATTATATTCAATATTAAAAACGACCTTGTTTCTAC T 95) -WSN_PB2_65 SEQ ID NO: 125 AGCGAAAGCAGGTCAATTATATTCAATATAGTGTCGAATAGTTTAAAAAC GACCTTGTTTCTACT 96) -WSN_PB2_59 SEQ ID NO: 126 AGCGAAAGCAGGTCAATTATATTCAATACGAATAGTTTAAAAACGACCTT GTTTCTACT 97) -WSN_PB2_62 SEQ ID NO: 127 AGCGAAAGCAGGTCAATTATATTCAATGTGTCGAATAGTTTAAAAACGAC CTTGTTTCTACT 98) -WSN_PB2_52 SEQ ID NO: 128 AGCGAAAGCAGGTCAATTATATTCATAGTTTAAAAACGACCTTGTTTCTA CT 99) -WSN_PB2_63 SEQ ID NO: 129 AGCGAAAGCAGGTCAATTATATTCATTAGTGTCGAATAGTTTAAAAACGA CCTTGTTTCTACT 100) -WSN_PB2_44 SEQ ID NO: 130 AGCGAAAGCAGGTCAATTATATTAAAAACGACCTTGTTTCTACT 101) -WSN_PB2_60 SEQ ID NO: 131 AGCGAAAGCAGGTCAATTATATTTAGTGTCGAATAGTTTAAAAACGACCT TGTTTCTACT 102) -WSN_PB2_46 SEQ ID NO: 132 AGCGAAAGCAGGTCAATAATAGTTTAAAAACGACCTTGTTTCTACT 103) WSN_PB2_51 SEQ ID NO: 133- AGCGAAAGCAGGTCAAGTGTCGAATAGTTTAAAAACGACCTTGTTTCTAC T 104) -WSN_PB1_73 SEQ ID NO: 134 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACTTTT CTAAAAAATGCCTTGTTTCTACT 105) -WSN_PB1_69 SEQ ID NO: 135 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACTTTT AAAATGCCTTGTTTCTACT 106) -WSN_PB1_75 SEQ ID NO: 136 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACTCTT CATGAAAAAATGCCTTGTTTCTACT 107) -WSN_PB1_65 SEQ ID NO: 137 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACAAAA TGCCTTGTTTCTACT 108) -WSN_PB1_64 SEQ ID NO: 138 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTAAAAAT GCCTTGTTTCTACT 109) -WSN_PB1_61 SEQ ID NO: 139 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTAAAATGCC TTGTTTCTACT 110) -WSN_PB1_73 SEQ ID NO: 140 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTGTCCTTCA TGAAAAAATGCCTTGTTTCTACT 111) -WSN_PB1_63 SEQ ID NO: 141 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCCATGAAAAAATG CCTTGTTTCTACT 112) -WSN_PB1_60 SEQ ID NO: 142 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCTGAAAAAATGCCT TGTTTCTACT 113) -WSN_PB1_80 SEQ ID NO: 143 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCTGAATTTAGCTTG TCCTTCATGAAAAAATGCCTTGTTTCTACT 114) -WSN_PB1_63 SEQ ID NO: 144 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCACCTTCATGAAAAAATG CCTTGTTTCTACT 115) -WSN_PB1_65 SEQ ID NO: 145 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAGTCCTTCATGAAAAAA TGCCTTGTTTCTACT 116) -WSN_PB1_58 SEQ ID NO: 146 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCCATGAAAAAATGCCTTG TTTCTACT 117) -WSN_PB1_52 SEQ ID NO: 147 AGCGAAAGCAGGCAAACCATTTGAATGGATGTAAAAATGCCTTGTTTCTA CT 118) -WSN_PB1_63 SEQ ID NO: 148 AGCGAAAGCAGGCAAACCATTTGAATGGACTTGTCCTTCATGAAAAAATG CCTTGTTTCTACT 119) -WSN_PB1_59 SEQ ID NO: 149 AGCGAAAGCAGGCAAACCATTTGAATGGGTCCTTCATGAAAAAATGCCTT GTTTCTACT 120) -WSN_PB1_52 SEQ ID NO: 150 AGCGAAAGCAGGCAAACCATTTGAATGCATGAAAAAATGCCTTGTTTCTA CT 121) -WSN_PB1_48 SEQ ID NO: 151 AGCGAAAGCAGGCAAACCATTTTCATGAAAAAATGCCTTGTTTCTACT 122) -WSN_PB1_61 SEQ ID NO: 152 AGCGAAAGCAGGCAAACCATTTTTTAGCTTGTCCTTCATGAAAAAATGCC TTGTTTCTACT 123) -WSN_PB1_57 SEQ ID NO: 153 AGCGAAAGCAGGCAAACCATTAGCTTGTCCTTCATGAAAAAATGCCTTGT TTCTACT 124) -WSN_PB1_60 SEQ ID NO: 154 AGCGAAAGCAGGCAAACTGAATTTAGCTTGTCCTTCATGAAAAAATGCCT TGTTTCTACT 125) -WSN_PB1_63 SEQ ID NO: 155 AGCGAAAGCAGGCAAACTAGTGAATTTAGCTTGTCCTTCATGAAAAAATG CCTTGTTTCTACT 126) -WSN_PB1_56 SEQ ID NO: 156 AGCGAAAGCAGGCAAAATTTAGCTTGTCCTTCATGAAAAAATGCCTTGTT TCTACT 127) -WSN_PA_63 SEQ ID NO: 157 AGCGAAAGCAGGTACTGATTCAAAATGGAAGATTTTGTTCCAAAAAAGTA CCTTGTTTCTACT 128) -WSN_PA_58 SEQ ID NO: 158 AGCGAAAGCAGGTACTGATTCAAAATGGAAGATTTTAAAAAAGTACCTTG TTTCTACT 129) -WSN_PA_53 SEQ ID NO: 159 AGCGAAAGCAGGTACTGATTCAAAATGGAAGATTAAAGTACCTTGTTTCT ACT 130) -WSN_PA_63 SEQ ID NO: 160 AGCGAAAGCAGGTACTGATTTATTTGCTATCCATACTGTCCAAAAAAGTA CCTTGTTTCTACT 131) -WSN_PA_44 SEQ ID NO: 161 AGCGAAAGCAGGTACTGAGTCCAAAAAAGTACCTTGTTTCTACT 132) -WSN_NS_59 SEQ ID NO: 162 AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTAACACCCTT GTTTCTACT 133) -WSN_NS_60 SEQ ID NO: 163 AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAATAAAAAACACCCT TGTTTCTACT 134) -WSN_NS_52 SEQ ID NO: 164 AGCAAAAGCAGGGTGACAAAGACATAATGTAAAAAACACCCTTGTTTCTA CT 135) -WSN_NS_48 SEQ ID NO: 165 AGCAAAAGCAGGGTGACAAAGACATTAAAAAACACCCTTGTTTCTACT 136) -WSN_NS_44 SEQ ID NO: 166 AGCAAAAGCAGGGTGACAAAGACAAAAACACCCTTGTTTCTACT 137) -WSN_NS_46 SEQ ID NO: 167 AGCAAAAGCAGGGTGACAAAGACAAAAAAACACCCTTGTTTCTACT 138) -WSN_NS_45 SEQ ID NO: 168 AGCAAAAGCAGGGTGACAAAGATAAAAAACACCCTTGTTTCTACT 139) -WSN_NS_67 SEQ ID NO: 169 AGCAAAAGCAGGGTGACAAAGTCTCGTTTCAGCTTATTTAATAATAAAAA ACACCCTTGTTTCTACT 140) -WSN_NS_46 SEQ ID NO: 170 AGCAAAAGCAGGGTGACAAATAATAAAAAACACCCTTGTTTCTACT 141) -WSN_NS_63 SEQ ID NO: 171 AGCAAAAGCAGGGTGACACTCGTTTCAGCTTATTTAATAATAAAAAACAC CCTTGTTTCTACT 142) -WSN_NP_94 SEQ ID NO: 172 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC GACCAAAGGCACCAAACGATCTTACAAATACCCTTGTTTCTACT 143) -WSN_NP_71 SEQ ID NO: 173 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC AAAAATACCCTTGTTTCTACT 144) -WSN_NP_74 SEQ ID NO: 174 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC AAGAAAAATACCCTTGTTTCTACT 145) -WSN_NP_76 SEQ ID NO: 175 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC TAAAGAAAAATACCCTTGTTTCTACT 146) -WSN_NP_91 SEQ ID NO: 176 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGA GAGGAGTACGACAATTAAAGAAAAATACCCTTGTTTCTACT 147) -WSN_NP_64 SEQ ID NO: 177 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATAAAATA CCCTTGTTTCTACT 148) -WSN_NP_65 SEQ ID NO: 178 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAAGAAAAAT ACCCTTGTTTCTACT 149) -WSN_NP_63 SEQ ID NO: 179 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAGAAAAATAC CCTTGTTTCTACT 150) -WSN_NP_88 SEQ ID NO: 180 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGCAATGCAGAG GAGTACGACAATTAAAGAAAAATACCCTTGTTTCTACT 151) -WSN_NP_60 SEQ ID NO: 181 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCAAAAATACCCT TGTTTCTACT 152) -WSN_NP_64 SEQ ID NO: 182 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCAAAGAAAAATA CCCTTGTTTCTACT 153) -WSN_NP_84 SEQ ID NO: 183 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATATGCAGAGGAGT ACGACAATTAAAGAAAAATACCCTTGTTTCTACT 154) -WSN_NP_59 SEQ ID NO: 184 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACAGAAAAATACCCTT GTTTCTACT 155) -WSN_NP_71 SEQ ID NO: 185 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACAACGACAATTAAAG AAAAATACCCTTGTTTCTACT 156) -WSN_NP_64 SEQ ID NO: 186 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGAAATTAAAGAAAAATA CCCTTGTTTCTACT 157) -WSN_NP_71 SEQ ID NO: 187 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAATAATAACAAAAC TCCTTGTTTCTACT 168) -WSN_NA_66 SEQ ID NO: 198 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGATAGTTTGTTCAAAAA ACTCCTTGTTTCTACT 169) -WSN_NA_67 SEQ ID NO: 199 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAAAGTAGTTTGTTCAAAA AACTCCTTGTTTCTACT 170) -WSN_NA_68 SEQ ID NO: 200 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCACAAGTAGTTTGTTCAAA AAACTCCTTGTTTCTACT 171) -WSN_NA_69 SEQ ID NO: 201 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAACAAGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 172) -WSN_NA_72 SEQ ID NO: 202 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCATTGACAAGTAGTTTGTT CAAAAAACTCCTTGTTTCTACT 173) -WSN_NA_63 SEQ ID NO: 203 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCTAGTTTGTTCAAAAAACT CCTTGTTTCTACT 174) -WSN_NA_69 SEQ ID NO: 204 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCGACAAGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 175) -WSN_NA_65 SEQ ID NO: 205 AGCAAAAGCAGGAGTTTAAATGAATCCAAACAAGTAGTTTGTTCAAAAAA CTCCTTGTTTCTACT  176)-WSN_NA_66 SEQ ID NO: 206 AGCAAAAGCAGGAGTTTAAATGAATCCAAAACAAGTAGTTTGTTCAAAAA ACTCCTTGTTTCTACT 177) -WSN_NA_67 SEQ ID NO: 207 AGCAAAAGCAGGAGTTTAAATGAATCCAAAGACAAGTAGTTTGTTCAAAA AACTCCTTGTTTCTACT 178) -WSN_NA_68 SEQ ID NO: 208 AGCAAAAGCAGGAGTTTAAATGAATCCAAATGACAAGTAGTTTGTTCAAA AAACTCCTTGTTTCTACT 179) -WSN_NA_65 SEQ ID NO: 209 AGCAAAAGCAGGAGTTTAAATGAATCCAGACAAGTAGTTTGTTCAAAAAA CTCCTTGTTTCTACT 180) -WSN_NA_68 SEQ ID NO: 210 AGCAAAAGCAGGAGTTTAAATGAATCCCATTGACAAGTAGTTTGTTCAAA AAACTCCTTGTTTCTACT 181) -WSN_NA_55 SEQ ID NO: 211 AGCAAAAGCAGGAGTTTAAATGAGTAGTTTGTTCAAAAAACTCCTTGTTT CTACT 182) -WSN_NA_63 SEQ ID NO: 212 AGCAAAAGCAGGAGTTTAAATGCATTGACAAGTAGTTTGTTCAAAAAACT CCTTGTTTCTACT 183) -WSN_NA_49 SEQ ID NO: 213 AGCAAAAGCAGGAGTTTAAATTTTGTTCAAAAAACTCCTTGTTTCTACT 184) -WSN_NA_56 SEQ ID NO: 214 AGCAAAAGCAGGAGTTTAAATCAAGTAGTTTGTTCAAAAAACTCCTTGTT TCTACT 185) -WSN_NA_43 SEQ ID NO: 215 AGCAAAAGCAGGAGTTTAATTCAAAAAACTCCTTGTTTCTACT 186) -WSN_NA_62 SEQ ID NO: 216 AGCAAAAGCAGGAGTTTACACCATTGACAAGTAGTTTGTTCAAAAAACTC CTTGTTTCTACT 187) -WSN_M_78 SEQ ID NO: 217 AGCGAAAGCAGGTAGATATTGAAAGATGAGTCTTCTAACCGAGGTCGAAA CGGAGTAAAAAACTACCTTGTTTCTACT 188) -WSN_M_61 SEQ ID NO: 218 AGCGAAAGCAGGTAGATATTGAAAGATTAGAGCTGGAGTAAAAAACTACC TTGTTTCTACT 189) -WSN_M_50 SEQ ID NO: 219 AGCGAAAGCAGGTAGATATTGAAAGATTAAAAAACTACCTTGTTTCTACT 190) -WSN_M_43 SEQ ID NO: 220 AGCGAAAGCAGGTAGATATTTAAAAAACTACCTTGTTTCTACT 191) -WSN_M_46 SEQ ID NO: 221 AGCGAAAGCAGGTAGATATGGAGTAAAAAACTACCTTGTTTCTACT 192) -WSN_HA_77 SEQ ID NO: 222 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGTAGGATTTCAGAAAT ATAAGGAAAAACACCCTTGTTTCTACT 193) -WSN_HA_53 SEQ ID NO: 223 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATAAACACCCTTGTTTCT ACT 194) -WSN_HA_61 SEQ ID NO: 224 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATATAAGGAAAAACACCC TTGTTTCTACT 195) -WSN_HA_65 SEQ ID NO: 225 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATAAATATAAGGAAAAAC ACCCTTGTTTCTACT 196) -WSN_HA_60 SEQ ID NO: 226 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAAATAAGGAAAAACACCCT TGTTTCTACT 197) -WSN_HA_87 SEQ ID NO: 227 AGCAAAAGCAGGGGAAAATAAAAACAACCAATATGCATCTGAGATTAGGA TTTCAGAAATATAAGGAAAAACACCCTTGTTTCTACT 198) -WSN_HA_65 SEQ ID NO: 228 AGCAAAAGCAGGGGAAAATAAAAACAACCATCAGAAATATAAGGAAAAAC ACCCTTGTTTCTACT 199) -WSN_HA_66 SEQ ID NO: 229 AGCAAAAGCAGGGGAAAATAAAAACAACCATTCAGAAATATAAGGAAAAA CACCCTTGTTTCTACT 200) -WSN_HA_51 SEQ ID NO: 230 AGCAAAAGCAGGGGAAAATAAAAACAACCGAAAAACACCCTTGTTTCTAC T 201) -WSN_HA_50 SEQ ID NO: 231 AGCAAAAGCAGGGGAAAATAAAAACAACGAAAAACACCCTTGTTTCTACT 202) -WSN_HA_46 SEQ ID NO: 232 AGCAAAAGCAGGGGAAAATAAAAACAAAAACACCCTTGTTTCTACT 203) -WSN_HA_65 SEQ ID NO: 233 AGCAAAAGCAGGGGAAAATAAAAACAGATTTCAGAAATATAAGGAAAAAC ACCCTTGTTTCTACT 204) -WSN_HA_44 SEQ ID NO: 234 AGCAAAAGCAGGGGAAAATAAAAAAAAACACCCTTGTTTCTACT 205) -WSN_HA_56 SEQ ID NO: 235 AGCAAAAGCAGGGGAAAATAAAAAGAAATATAAGGAAAAACACCCTTGTT TCTACT 206) -WSN_HA_60 SEQ ID NO: 236 AGCAAAAGCAGGGGAAAATAAAAATTCAGAAATATAAGGAAAAACACCCT TGTTTCTACT 207) -WSN_HA_63 SEQ ID NO: 237 AGCAAAAGCAGGGGAAAATAAAAAGATTTCAGAAATATAAGGAAAAACAC CCTTGTTTCTACT 208) -WSN_HA_44 SEQ ID NO: 238 AGCAAAAGCAGGGGAAAATAAGGAAAAACACCCTTGTTTCTACT 209) -WSN_HA_60 SEQ ID NO: 239 AGCAAAAGCAGGGGAAAATAGGATTTCAGAAATATAAGGAAAAACACCCT TGTTTCTACT 210) -WSN_HA_53 SEQ ID NO: 240 AGCAAAAGCAGGGGAAAATCAGAAATATAAGGAAAAACACCCTTGTTTCT ACT 211) -WSN_HA_58 SEQ ID NO: 241 AGCAAAAGCAGGGGAAAATGATTTCAGAAATATAAGGAAAAACACCCTTG TTTCTACT 212) -WSN_HA_41 SEQ ID NO: 242 AGCAAAAGCAGGGGAAAAGGAAAAACACCCTTGTTTCTACT 213) -WSN_HA_43 SEQ ID NO: 243 AGCAAAAGCAGGGGAAAAAAGGAAAAACACCCTTGTTTCTACT 214) -WSN_HA_50 SEQ ID NO: 244 AGCAAAAGCAGGGGAAAAGAAATATAAGGAAAAACACCCTTGTTTCTACT 215) -WSN_HA_64 SEQ ID NO: 245 AGCAAAAGCAGGGGAAAAAGATTAGGATTTCAGAAATATAAGGAAAAACA CCCTTGTTTCTACT

1) WSN_NP_61 SEQ ID NO: 246 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATAGAAAAATACCC TTGTTTCTACT 2) WSN_PB1_57 SEQ ID NO: 247 AGCGAAAGCAGGCAAACCATTTGAATGGATTTCATGAAAAAATGCCTTGT TTCTACT 3) WSN_PB1_57 SEQ ID NO: 248 AGCGAAAGCAGGCAAACCATTTGAATGTCCTTCATGAAAAAATGCCTTGT TTCTACT 4) WSN_PB1_62 SEQ ID NO: 249 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTAAAATGC CTTGTTTCTACT 5) WSN_PB1_66 SEQ ID NO: 250 AGCGAAAGCAGGCAAACCATTTGAATGTTTAGCTTGTCCTTCATGAAAAA ATGCCTTGTTTCTACT 6) WSN_NP_58 SEQ ID NO: 251 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCAAATACCCTTG TTTCTACT 7) WSN_HA_60 SEQ ID NO: 252 AGCAAAAGCAGGGGAAATTAGGATTTCAGAAATATAAGGAAAAACACCCT TGTTTCTACT 8) WSN_HA_44 SEQ ID NO: 253 AGCAAAAGCAGGGGAATATAAGGAAAAACACCCTTGTTTCTACT 9) WSN_PB1_69 SEQ ID NO: 254 AGCGAAAGCAGGCAAACCATTTGAATGGATGTTAGCTTGTCCTTCATGAA AAAATGCCTTGTTTCTACT 10) WSN_PB1_61 SEQ ID NO: 255 AGCGAAAGCAGGCAAACCATTTGAATGGTTGTCCTTCATGAAAAAATGCC TTGTTTCTACT 11) WSN_NP_63 SEQ ID NO: 256 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACTTAAAGAAAAATAC CCTTGTTTCTACT 12) WSN_NA_70 SEQ ID NO: 257 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCTGACAAGTAGTTTGTTCA AAAAACTCCTTGTTTCTACT 13) WSN_NA_62 SEQ ID NO: 258 AGCAAAAGCAGGAGTTTACACCATTGACAAGTAGTTTGTTCAAAAAACTC CTTGTTTCTACT 14) WSN_PB1_65 SEQ ID NO: 259 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTGAAAAAA TGCCTTGTTTCTACT 15) WSN_PB1_69 SEQ ID NO: 260 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGTCCTTCATGAA AAAATGCCTTGTTTCTACT 16) WSN_PB1_48 SEQ ID NO: 261 AGCGAAAGCAGGCAAACCATTTGAATGAAAAAATGCCTTGTTTCTACT 17) WSN_PB1_62 SEQ ID NO: 262 AGCGAAAGCAGGCAAACCATTTGAATAGCTTGTCCTTCATGAAAAAATGC CTTGTTTCTACT 18) WSN_NP_71 SEQ ID NO: 263 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC AAAAATACCCTTGTTTCTACT 19) WSN_NP_76 SEQ ID NO: 264 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATCATGGC TAAAGAAAAATACCCTTGTTTCTACT 20) WSN_NP_64 SEQ ID NO: 265 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGAAATAAAATA CCCTTGTTTCTACT 21) WSN_NP_55 SEQ ID NO: 266 AGCAAAAGCAGGGTAGATAATCACTCACAGAGAGAAAAATACCCTTGTTT CTACT 22) WSN_NP_44 SEQ ID NO: 267 AGCAAAAGCAGGGTAGATTAAAGAAAAATACCCTTGTTTCTACT 23) WSN_HA 58 SEQ ID NO: 268 AGCAAAAGCAGGGGAATAGGATTTCAGAAATATAAGGAAAAACACCCTTG TTTCTACT 24) WSN_HA 64 SEQ ID NO: 269 AGCAAAAGCAGGGGAATGAGATTAGGATTTCAGAAATATAAGGAAAAACA CCCTTGTTTCTACT 25) WSN_PB2_80 SEQ ID NO: 270 AGCGAAAGCAGGTCAATTATATTCAATATGGAAGGCCATCAATTAGTGTC GAATAGTTTAAAAACGACCTTGTTTCTACT 26) WSN_PB1_77 SEQ ID NO: 271 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACTTTT CTCATGAAAAAATGCCTTGTTTCTACT 27) WSN_PB1_65 SEQ ID NO: 272 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACAAAA TGCCTTGTTTCTACT 28) WSN_PB1_64 SEQ ID NO: 273 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTAAAAAAT GCCTTGTTTCTACT 29) WSN_PB1_68 SEQ ID NO: 274 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTCATGAAA AAATGCCTTGTTTCTACT 30) WSN_PB1_64 SEQ ID NO: 275 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTGAAAAAAT GCCTTGTTTCTACT 31) WSN_PB1_58 SEQ ID NO: 276 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCAAAAATGCCTTG TTTCTACT 32) WSN_PB1_64 SEQ ID NO: 277 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCTCATGAAAAAAT GCCTTGTTTCTACT 33) WSN_PB1_68 SEQ ID NO: 278 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCCTCCTTCATGAAA AAATGCCTTGTTTCTACT 34) WSN_PB1_60 SEQ ID NO: 279 AGCGAAAGCAGGCAAACCATTTGAATGGATGTCAATCTGAAAAAATGCCT TGTTTCTACT 35) WSN_PB1_54 SEQ ID NO: 280 AGCGAAAGCAGGCAAACCATTTGAATGGATGTGAAAAAATGCCTTGTTTC TACT  36) WSN_PB1_50 SEQ ID NO: 281 AGCGAAAGCAGGCAAACCATTTGAATGGGAAAAAATGCCTTGTTTCTACT 37) WSN_PB1_66 SEQ ID NO: 282 AGCGAAAGCAGGCAAACCATTTGAATGGTTAGCTTGTCCTTCATGAAAAA ATGCCTTGTTTCTACT 38) WSN_PB1_49 SEQ ID NO: 283 AGCGAAAGCAGGCAAACCATTTGAATTGAAAAAATGCCTTGTTTCTACT  39) WSN_PB1_48 SEQ ID NO: 284 AGCGAAAGCAGGCAAACCATTTGCATGAAAAAATGCCTTGTTTCTACT 40) WSN_PB1_43 SEQ ID NO: 285 AGCGAAAGCAGGCAAACCATTTAAAAAATGCCTTGTTTCTACT 41) WSN_PB1_64 SEQ ID NO: 286 AGCGAAAGCAGGCAAACCATGTGAATTTAGCTTGTCCTTCATGAAAAAAT GCCTTGTTTCTACT 42) WSN_PB1_61 SEQ ID NO: 287 AGCGAAAGCAGGCAAACCTGAATTTAGCTTGTCCTTCATGAAAAAATGCC TTGTTTCTACT 43) WSN_PB1_40 SEQ ID NO: 288 AGCGAAAGCAGGCAAACTGAAAAAATGCCTTGTTTCTACT 44) WSN_PB1_56 SEQ ID NO: 289 AGCGAAAGCAGGCAAACTTTAGCTTGTCCTTCATGAAAAAATGCCTTGTT TCTACT 45) WSN_PA_60 SEQ ID NO: 290 AGCGAAAGCAGGTACTGATTCAAAATGGCATACTGTCCAAAAAAGTACCT TGTTTCTACT 46) WSN_PA_66 SEQ ID NO: 291 AGCGAAAGCAGGTACTGATTCAAAATGTGCTATCCATACTGTCCAAAAAA GTACCTTGTTTCTACT 47) WSN_NS_80 SEQ ID NO: 292 AGCAAAAGCAGGGTGACAAAGACATAATGGATCCAAACACTGTGTCAAGC TTTCAGATAAAAAACACCCTTGTTTCTACT 48) WSN_NS 56 SEQ ID NO: 293 AGCAAAAGCAGGGTGACAAAGACATAATGGATCTAAAAAACACCCTTGTT TCTACT 49) WSN_NS_55 SEQ ID NO: 294 AGCAAAAGCAGGGTGACAAAGACATAATGTAATAAAAAACACCCTTGTTT CTACT 50) WSN_NP_66 SEQ ID NO: 295 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCGTAAAGAAAAA TACCCTTGTTTCTACT 51) WSN_NP_60 SEQ ID NO: 296 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCAAAAATACCCT TGTTTCTACT 52) WSN_NP_65 SEQ ID NO: 297 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCTAAAGAAAAAT ACCCTTGTTTCTACT 53) WSN_NP_66 SEQ ID NO: 298 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCTTAAAGAAAAA TACCCTTGTTTCTACT 54) WSN_NP_74 SEQ ID NO: 299 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATCTACGACAATTA AAGAAAAATACCCTTGTTTCTACT 55) WSN_NP_64 SEQ ID NO: 300 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACATTAAAGAAAAATA CCCTTGTTTCTACT 56) WSN_NP_59 SEQ ID NO: 301 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACAGAAAAATACCCTT GTTTCTACT 57) WSN_NP_72 SEQ ID NO: 302 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGACGTACGACAATTAAA GAAAAATACCCTTGTTTCTACT 58) WSN_NP_70 SEQ ID NO: 303 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTGATACGACAATTAAAGA AAAATACCCTTGTTTCTACT 59) WSN_NP_57 SEQ ID NO: 304 AGCAAAAGCAGGGTAGATAATCACTCACAGAGTAAGAAAAATACCCTTGT TTCTACT 60) WSN_NP_44 SEQ ID NO: 305 AGCAAAAGCAGGGTAGATAATCACTAAATACCCTTGTTTCTACT 61) WSN_NP_49 SEQ ID NO: 306 AGCAAAAGCAGGGTAGATAATCACTAAGAAAAATACCCTTGTTTCTACT 62) WSN_NP_43 SEQ ID NO: 307 AGCAAAAGCAGGGTAGTTAAAGAAAAATACCCTTGTTTCTACT 63) WSN_NA_75 SEQ ID NO: 308 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAATAAAAGTAGTTT GTTCAAAAAACTCCTTGTTTCTACT 64) WSN_NA_73 SEQ ID NO: 309 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAACAAGTAGTTTGT TCAAAAAACTCCTTGTTTCTACT 65) WSN_NA_79 SEQ ID NO: 310 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAACATTGACAAGTA GTTTGTTCAAAAAACTCCTTGTTTCTACT 66) WSN_NA_69 SEQ ID NO: 311 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGAAAGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 67) WSN_NA_61 SEQ ID NO: 312 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGATGTTCAAAAAACTCC TTGTTTCTACT 68) WSN_NA 60 SEQ ID NO: 313 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAGTGTTCAAAAAACTCCT TGTTTCTACT 69) WSN_NA_69 SEQ ID NO: 314 AGCAAAAGCAGGAGTTTAAATGAATCCAAACCAACAAGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 70) WSN_NA_66 SEQ ID NO: 315 AGCAAAAGCAGGAGTTTAAATGAATCCAAAACAAGTAGTTTGTTCAAAAA ACTCCTTGTTTCTACT 71) WSN_NA_60 SEQ ID NO: 316 AGCAAAAGCAGGAGTTTAAATGAATCCAATAGTTTGTTCAAAAAACTCCT TGTTTCTACT 72) WSN_NA_63 SEQ ID NO: 317 AGCAAAAGCAGGAGTTTAACACCATTGACAAGTAGTTTGTTCAAAAAACT CCTTGTTTCTACT 73) WSN_NA_62 SEQ ID NO: 318 AGCAAAAGCAGGAGTTTTCACCATTGACAAGTAGTTTGTTCAAAAAACTC CTTGTTTCTACT 74) WSN_NA_63 SEQ ID NO: 319 AGCAAAAGCAGGAGTCGTTCACCATTGACAAGTAGTTTGTTCAAAAAACT CCTTGTTTCTACT 75) WSN_M_71 SEQ ID NO: 320 AGCGAAAGCAGGTAGATATTGAAAGATGAGTCTTCCATAGAGCTGGAGTA AAAAACTACCTTGTTTCTACT 76) WSN_M_52 SEQ ID NO: 321 AGCGAAAGCAGGTAGATATTGAAAGATGAGAAAAAACTACCTTGTTTCTA CT 77) WSN_M_48 SEQ ID NO: 322 AGCGAAAGCAGGTAGATATTGAAAGATAAAAACTACCTTGTTTCTACT 78) WSN_M 47 SEQ ID NO: 323 AGCGAAAGCAGGTAGATATTGAAATAAAAAACTACCTTGTTTCTACT 79) WSN_HA_89 SEQ ID NO: 324 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGAAGGCAGATTAGGAT TTCAGAAATATAAGGAAAAACACCCTTGTTTCTACT 80) WSN_HA_90 SEQ ID NO: 325 AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGAAGGCCTGAGATTAG GATTTCAGAAATATAAGGAAAAACACCCTTGTTTCTACT 81) WSN_HA_50 SEQ ID NO: 326 AGCAAAAGCAGGGGAAAATAAAAACAACGAAAAACACCCTTGTTTCTACT 82) WSN_HA_63 SEQ ID NO: 327 AGCAAAAGCAGGGGAAAATAAAAACATTTCAGAAATATAAGGAAAAACAC CCTTGTTTCTACT 83) WSN_HA 44 SEQ ID NO: 328 AGCAAAAGCAGGGGAAAATAAAGAAAAACACCCTTGTTTCTACT 84) WSN_HA_41 SEQ ID NO: 329 AGCAAAAGCAGGGGAAAATGAAAAACACCCTTGTTTCTACT 85) WSN_HA_65 SEQ ID NO: 330 AGCAAAAGCAGGGGAAATGAGATTAGGATTTCAGAAATATAAGGAAAAAC ACCCTTGTTTCTACT 86) WSN_HA_66 SEQ ID NO: 331 AGCAAAAGCAGGGGAAACTGAGATTAGGATTTCAGAAATATAAGGAAAAA CACCCTTGTTTCTACT 87) WSN_HA_61 SEQ ID NO: 332 AGCAAAAGCAGGGGAAGATTAGGATTTCAGAAATATAAGGAAAAACACCC TTGTTTCTACT

1) SEQ ID NO: 333 BM18_PB2, 1, 2325, 35: (18+17) AGCRAAAGCAGGTCAATTACGACCTTGTTTCTACT 2) SEQ ID NO: 334 BM18_M, 1, 1009, 36: (17+19) AGCRAAAGCAGGTAGATAAACTACCTTGTTTCTACT 3) SEQ ID NO: 335 BM18_M, 1, 1010, 35: (17+18) AGCRAAAGCAGGTAGATAACTACCTTGTTTCTACT 4) SEQ ID NO: 336 BM18_PB2, 1, 2324, 36: (18+18) AGCRAAAGCAGGTCAATTAACGACCTTGTTTCTACT 5) SEQ ID NO: 337 BM18_NS, 1, 872, 36: (17+19) AGCRAAAGCAGGGTGACAAACACCCTTGTTTCTACT 6) SEQ ID NO: 338 BM18_NP, 1, 1547, 37: (18+19) AGCRAAAGCAGGGTAGATAAATACCCTTGTTTCTACT 7) SEQ ID NO: 339 BM18_HA, 1, 1762, 36: (19+17) AGCRAAAGCAGGGGAAAATACACCCTTGTTTCTACT 8) SEQ ID NO: 340 BM18_NP, 1, 1541, 41: (16+25) AGCRAAAGCAGGGTAGAAAGAAAAATACCCTTGTTTCTACT 9) SEQ ID NO: 341 BM18_NP, 1, 1547, 35: (16+19) AGCRAAAGCAGGGTAGAAATACCCTTGTTTCTACT 10) SEQ ID NO: 342 BM18_PB2, 1, 2323, 37: (18+19) AGCRAAAGCAGGTCAATTAAACGACCTTGTTTCTACT 11) SEQ ID NO: 343 BM18_PB2, 1, 2319, 39: (16+23) AGCRAAAGCAGGTCAATTAAAAACGACCTTGTTTCTACT 12) SEQ ID NO: 344 BM18_HA, 1, 1761, 37: (19+18) AGCRAAAGCAGGGGAAAATAACACCCTTGTTTCTACT 13) SEQ ID NO: 345 BM18_NS, 1, 869, 39: (17+22) AGCRAAAGCAGGGTGACAAAAAACACCCTTGTTTCTACT 14) SEQ ID NO: 346 BM18_NP, 1, 1545, 39: (18+21) AGCRAAAGCAGGGTAGATAAAAATACCCTTGTTTCTACT 15) SEQ ID NO: 347 BM18_NP, 1, 1548, 34: (16+18) AGCRAAAGCAGGGTAGAATACCCTTGTTTCTACT 16) SEQ ID NO: 348 BM18_M, 1, 1008, 37: (17+20) AGCRAAAGCAGGTAGATAAAACTACCTTGTTTCTACT 17) SEQ ID NO: 349 BM18_NS, 1, 871, 37: (17+20) AGCRAAAGCAGGGTGACAAAACACCCTTGTTTCTACT 18) SEQ ID NO: 350 BM18_NS, 1, 873, 35: (17+18) AGCRAAAGCAGGGTGACAACACCCTTGTTTCTACT 19) SEQ ID NO: 351 BM18_HA, 1, 1760, 38: (19+19) AGCRAAAGCAGGGGAAAATAAACACCCTTGTTTCTACT 20) SEQ ID NO: 352 BM18_HA, 1, 1752, 44: (17+27) AGCRAAAGCAGGGGAAAATAAGGAAAAACACCCTTGTTTCTACT 21) SEQ ID NO: 353 BM18_NP, 1, 1546, 36: (16+20) AGCRAAAGCAGGGTAGAAAATACCCTTGTTTCTACT 22) SEQ ID NO: 354 BM18_M, 1, 1005, 39: (16+23) AGCRAAAGCAGGTAGATAAAAAACTACCTTGTTTCTACT 23) SEQ ID NO: 355 BM18_NP, 1, 1546, 38: (18+20) AGCRAAAGCAGGGTAGATAAAATACCCTTGTTTCTACT 24) SEQ ID NO: 356 BM18_PB1, 1, 2324, 36: (18+18) AGCRAAAGCAGGCAAACCAAATGCCTTGTTTCTACT 25) SEQ ID NO: 357 BM18_HA, 1, 1757, 41: (19+22) AGCRAAAGCAGGGGAAAATGAAAAACACCCTTGTTTCTACT 26) SEQ ID NO: 358 BM18_NP, 1, 1540, 43: (17+26) AGCRAAAGCAGGGTAGATAAAGAAAAATACCCTTGTTTCTACT 27) SEQ ID NO: 359 BM18_NP, 1, 1544, 38: (16+22) AGCRAAAGCAGGGTAGGAAAAATACCCTTGTTTCTACT 28) SEQ ID NO: 360 BM18_PB1, 1, 2323, 37: (18+19) AGCRAAAGCAGGCAAACCAAAATGCCTTGTTTCTACT 29) SEQ ID NO: 361 BM18_HA, 1, 1758, 37: (16+21) AGCRAAAGCAGGGGAAAAAAACACCCTTGTTTCTACT 30) SEQ ID NO: 362 BM18_NP, 1, 1549, 35: (18+17) AGCRAAAGCAGGGTAGATATACCCTTGTTTCTACT 31) SEQ ID NO: 363 BM18_PB2, 1, 2322, 38: (18+20) AGCRAAAGCAGGTCAATTAAAACGACCTTGTTTCTACT 32) SEQ ID NO: 364 BM18_M, 1, 1011, 34: (17+17) AGCRAAAGCAGGTAGATACTACCTTGTTTCTACT 33) SEQ ID NO: 365 BM18_HA, 1, 1759, 39: (19+20) AGCRAAAGCAGGGGAAAATAAAACACCCTTGTTTCTACT 34) SEQ ID NO: 366 BM18_PB2, 1, 2315, 44: (17+27) AGCRAAAGCAGGTCAATTAGTTTAAAAACGACCTTGTTTCTACT 35) SEQ ID NO: 367 BM18_NP, 1, 1543, 39: (16+23) AGCRAAAGCAGGGTAGAGAAAAATACCCTTGTTTCTACT 36) SEQ ID NO: 368 BM18_HA, 1, 1758, 38: (17+21) AGCRAAAGCAGGGGAAAAAAAACACCCTTGTTTCTACT 37) SEQ ID NO: 369 BM18_HA, 1, 1754, 42: (17+25) AGCRAAAGCAGGGGAAAAAGGAAAAACACCCTTGTTTCTACT 38) SEQ ID NO: 370 BM18_PB1, 1, 2325, 35: (18+17) AGCRAAAGCAGGCAAACCAATGCCTTGTTTCTACT 39) SEQ ID NO: 371 BM18_NS, 1, 867, 47: (23+24) AGCRAAAGCAGGGTGACAAAGACATAAAAAACACCCTTGTTTCTACT 40) SEQ ID NO: 372 BM18_NS, 1, 870, 38: (17+21) AGCRAAAGCAGGGTGACAAAAACACCCTTGTTTCTACT 41) SEQ ID NO: 373: BM18_NS, 1, 874, 34: (17+17) AGCRAAAGCAGGGTGACACACCCTTGTTTCTACT 42) SEQ ID NO: 374 BM18_PB1, 1, 2321, 39: (18+21) AGCRAAAGCAGGCAAACCAAAAAATGCCTTGTTTCTACT 43) SEQ ID NO: 375 BM18_M, 1, 1007, 38: (17+21) AGCRAAAGCAGGTAGATAAAAACTACCTTGTTTCTACT 44) SEQ ID NO: 376 BM18_HA, 1, 1756, 42: (19+23) AGCRAAAGCAGGGGAAAATGGAAAAACACCCTTGTTTCTACT 45) SEQ ID NO: 377 BM18_NA, 1, 1439, 36: (17+19) AGCRAAAGCAGGAGTTTAAAACTCCTTGTTTCTACT 46) SEQ ID NO: 378 BM18_PB1, 1, 2322, 38: (18+20) AGCRAAAGCAGGCAAACCAAAAATGCCTTGTTTCTACT 47) SEQ ID NO: 379 BM18_M, 1, 1004, 41: (17+24) AGCRAAAGCAGGTAGATGTAAAAAACTACCTTGTTTCTACT 48) SEQ ID NO: 380 BM18_NP, 1, 1544, 40: (18+22) AGCRAAAGCAGGGTAGATGAAAAATACCCTTGTTTCTACT 49) SEQ ID NO: 381 BM18_NP, 1, 1542, 40: (16+24) AGCRAAAGCAGGGTAGAAGAAAAATACCCTTGTTTCTACT 50) SEQ ID NO: 382 BM18_HA, 1, 1754, 41: (16+25) AGCRAAAGCAGGGGAAAAGGAAAAACACCCTTGTTTCTACT 51) SEQ ID NO: 383 BM18_PB2, 1, 2313, 47: (18+29) AGCRAAAGCAGGTCAATTAATAGTTTAAAAACGACCTTGTTTCTACT 52) SEQ ID NO: 384 BM18_M, 1, 1005, 42: (19+23) AGCRAAAGCAGGTAGATGTTAAAAAACTACCTTGTTTCTACT 53) SEQ ID NO: 385 BM18_NP, 1, 1543, 41: (18+23) AGCRAAAGCAGGGTAGATAGAAAAATACCCTTGTTTCTACT 54) SEQ ID NO: 386 BM18_PB2, 1, 2313, 53: (24+29) AGCRAAAGCAGGTCAATTATATTCAATAGTTTAAAAACGACCTTGTTT CTACT 55) SEQ ID NO: 387 BM18_PB2, 1, 2314, 46: (18+28) AGCRAAAGCAGGTCAATTATAGTTTAAAAACGACCTTGTTTCTACT 56) SEQ ID NO: 388 BM18_PB1, 1, 2322, 43: (23+20) AGCRAAAGCAGGCAAACCATTTGAAAAATGCCTTGTTTCTACT 57) SEQ ID NO: 389 BM18_PB1, 1, 2319, 44: (21+23) AGCRAAAGCAGGCAAACCATTTGAAAAAATGCCTTGTTTCTACT 58) SEQ ID NO: 390 BM18_M, 1, 1006, 43: (21+22) AGCRAAAGCAGGTAGATGTTGAAAAAACTACCTTGTTTCTACT 59) SEQ ID NO: 391 BM18_PA, 1, 2212, 48: (26+22) AGCRAAAGCAGGTACTGATTCAAAATAAAAAAGTACCTTGTTTCTACT 60) SEQ ID NO: 392 BM18_PA, 1, 2216, 39: (21+18) AGCRAAAGCAGGTACTGATTCAAGTACCTTGTTTCTACT 61) SEQ ID NO: 393 BM18_PA, 1, 2216, 34: (16+18) AGCRAAAGCAGGTACTAAGTACCTTGTTTCTACT 62) SEQ ID NO: 394 BM18_PA, 1, 2215, 35: (16+19) AGCRAAAGCAGGTACTAAAGTACCTTGTTTCTACT 63) SEQ ID NO: 395 BM18_PB2, 1, 2308, 60: (26+34) AGCRAAAGCAGGTCAATTATATTCAATGTCGAATAGTTTAAAAACGA CCTTGTTTCTACT 64) SEQ ID NO: 396 BM18_HA, 1, 1746, 52: (19+33) AGCRAAAGCAGGGGAAAATAGAAATATAAGGAAAAACACCCTTGTT TCTACT 65) SEQ ID NO: 397 BM18_HA, 1, 1741, 57: (19+38) AGCRAAAGCAGGGGAAAATATTTCAGAAATATAAGGAAAAACACCC TTGTTTCTACT 66) SEQ ID NO: 398 BM18_HA, 1, 1758, 39: (18+21) AGCRAAAGCAGGGGAAAAAAAAACACCCTTGTTTCTACT 67) SEQ ID NO: 399 BM18_HA, 1, 1737, 60: (18+42) AGCRAAAGCAGGGGAAAATAGGATTTCAGAAATATAAGGAAAAACA CCCTTGTTTCTACT 68) SEQ ID NO: 400 BM18_NS, 1, 869, 43: (21+22) AGCRAAAGCAGGGTGACAAAGAAAAAACACCCTTGTTTCTACT 69) SEQ ID NO: 401 BM18_M, 1, 1000, 46: (18+28) AGCRAAAGCAGGTAGATGTGGAGTAAAAAACTACCTTGTTTCTACT 70) SEQ ID NO: 402 BM18_NA, 1, 1409, 66: (17+49) AGCRAAAGCAGGAGTTTCCATTCACCATTGACAAGTAGTTTGTTCA AAAAACTCCTTGTTTCTACT 71) SEQ ID NO: 403 BM18_NA, 1, 1423, 51: (16+35) AGCRAAAGCAGGAGTTCAAGTAGTTTGTTCAAAAAACTCCTTGTTTC TACT 72) SEQ ID NO: 404 BM18_NP, 1, 1545, 38: (17+21) AGCRAAAGCAGGGTAGAAAAAATACCCTTGTTTCTACT 73) SEQ ID NO: 405 BM18_NP, 1, 1541, 42: (17+25) AGCRAAAGCAGGGTAGAAAAGAAAAATACCCTTGTTTCTACT 74) SEQ ID NO: 406 BM18_PA, 1, 2207, 52: (25+27) AGCRAAAGCAGGTACTGATTCAAAATGTCCAAAAAAGTACCTTG TTTCTACT 75) SEQ ID NO: 407 BM18_PA, 1, 2211, 43: (20+23) AGCRAAAGCAGGTACTGATTCAAAAAAGTACCTTGTTTCTACT 76) SEQ ID NO: 408 BM18_PB1, 1, 2320, 45: (23+22) AGCRAAAGCAGGCAAACCATTTGGAAAAAATGCCTTGTTTCTACT 77) SEQ ID NO: 409 BM18_PB1, 1, 2302, 58: (18+40) AGCRAAAGCAGGCAAACCATTTAGCTTGTCCTTCATGAAAAAATG CCTTGTTTCTACT 78) SEQ ID NO: 410 BM18_HA, 1, 1755, 43: (19+24) AGCRAAAGCAGGGGAAAATAGGAAAAACACCCTTGTTTCTACT 79) SEQ ID NO: 411 BM18_HA, 1, 1757, 38: (16+22) AGCRAAAGCAGGGGAAGAAAAACACCCTTGTTTCTACT 80) SEQ ID NO: 412 BM18_NS, 1, 864, 50: (23+27) AGCRAAAGCAGGGTGACAAAGACATAATAAAAAACACCCTTGTTTC TACT 81) SEQ ID NO: 413 BM18_NS, 1, 869, 42: (20+22) AGCRAAAGCAGGGTGACAAAAAAAAACACCCTTGTTTCTACT 82) SEQ ID NO: 414 BM18_M, 1, 988, 73: (33+40) AGCRAAAGCAGGTAGATGTTGAAAGATGAGTCTTCAACATAGAGCTGGA GTAAAAAACTACCTTGTTTCTACT 83) SEQ ID NO: 415 BM18_NA, 1, 1428, 62: (32+30) AGCRAAAGCAGGAGTTTAAATGAATCCAAATCAGTTTGTTCAAAAAACT CCTTGTTTCTACT 84) SEQ ID NO: 416 BM18_NA, 1, 1437, 38: (17+21) AGCRAAAGCAGGAGTTTAAAAAACTCCTTGTTTCTACT 85) SEQ ID NO: 417 BM18_NP, 1, 1547, 58: (39+19) AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAATACCCTTG TTTCTACT 86) SEQ ID NO: 418 BM18_NP, 1, 1546, 56: (36+20) AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACAAAATACCCTTGTT TCTACT 87) SEQ ID NO: 419 BM18_NP, 1, 1535, 59: (28+31) AGCRAAAGCAGGGTAGATAATCACTCACACAATTAAAGAAAAATACCCTT GTTTCTACT 88) SEQ ID NO: 420 BM18_NP, 1, 1540, 42: (16+26) AGCRAAAGCAGGGTAGTAAAGAAAAATACCCTTGTTTCTACT 89) SEQ ID NO: 421 BM18_PA, 1, 2213, 42: (21+21) AGCRAAAGCAGGTACTGATTCAAAAAGTACCTTGTTTCTACT 90) SEQ ID NO: 422 BM18_PA, 1, 2216, 35: (17+18) AGCRAAAGCAGGTACTGAAGTACCTTGTTTCTACT 91) SEQ ID NO: 423 BM18_PA, 1, 2217, 33: (16+17) AGCRAAAGCAGGTACTAGTACCTTGTTTCTACT 92) SEQ ID NO: 424 BM18_PB2, 1, 2312, 48: (18+30) AGCRAAAGCAGGTCAATTGAATAGTTTAAAAACGACCTTGTTTCTACT 93) SEQ ID NO: 425 BM18_PB2, 1, 2308, 51: (17+34) AGCRAAAGCAGGTCAATTGTCGAATAGTTTAAAAACGACCTTGTTTCT ACT 94) SEQ ID NO: 426 BM18_PB1, 1, 2296, 71: (25+46) AGCRAAAGCAGGCAAACCATTTGAATAGTGAATTTAGCTTGTCCTTCA TGAAAAAATGCCTTGTTTCTACT 95) SEQ ID NO: 427 BM18_PB1, 1, 2324, 40: (22+18) AGCRAAAGCAGGCAAACCATTTAAATGCCTTGTTTCTACT 96) SEQ ID NO: 428 BM18_PB1, 1, 2321, 43: (22+21) AGCRAAAGCAGGCAAACCATTTAAAAAATGCCTTGTTTCTACT 97) SEQ ID NO: 429 BM18_HA, 1, 1758, 46: (25+21) AGCRAAAGCAGGGGAAAATAAAAACAAAAACACCCTTGTTTCTACT 98) SEQ ID NO: 430 BM18_HA, 1, 1746, 55: (22+33) AGCRAAAGCAGGGGAAAATAAAAGAAATATAAGGAAAAACACCCTTGT TTCTACT 99) SEQ ID NO: 431 BM18_HA, 1, 1731, 66: (18+48) AGCRAAAGCAGGGGAAAATGAGATTAGGATTTCAGAAATATAAGGAAA AACACCCTTGTTTCTACT 100) SEQ ID NO: 432 BM18_HA, 1, 1757, 39: (17+22) AGCRAAAGCAGGGGAAAGAAAAACACCCTTGTTTCTACT 101) SEQ ID NO: 433 BM18_NS, 1, 873, 43: (25+18) AGCRAAAGCAGGGTGACAAAGACATAACACCCTTGTTTCTACT 102) SEQ ID NO: 434 BM18_NS, 1, 870, 46: (25+21) AGCRAAAGCAGGGTGACAAAGACATAAAAACACCCTTGTTTCTACT 103) SEQ ID NO: 435 BM18_NS, 1, 858, 57: (24+33) AGCRAAAGCAGGGTGACAAAGACATATTTAATAATAAAAAACACCCT TGTTTCTACT 104) SEQ ID NO: 436 BM18_NS, 1, 872, 42: (23+19) AGCRAAAGCAGGGTGACAAAGACAAACACCCTTGTTTCTACT 105) SEQ ID NO: 437 BM18_NS, 1, 873, 39: (21+18) AGCRAAAGCAGGGTGACAAAGAACACCCTTGTTTCTACT 106) SEQ ID NO: 438 BM18_NS, 1, 863, 49: (21+28) AGCRAAAGCAGGGTGACAAAGAATAATAAAAAACACCCTTGTTTC TACT 107) SEQ ID NO: 439 BM18_NS, 1, 866, 43: (18+25) AGCRAAAGCAGGGTGACAAATAAAAAACACCCTTGTTTCTACT 108) SEQ ID NO: 440 BM18_NS, 1, 866, 42: (17+25) AGCRAAAGCAGGGTGACAATAAAAAACACCCTTGTTTCTACT 109) SEQ ID NO: 441 BM18_M, 1, 1003, 56: (31+25) AGCRAAAGCAGGTAGATGTTGAAAGATGAGTAGTAAAAAACTACCTT GTTTCTACT 110) SEQ ID NO: 442 BM18_M, 1, 1008, 48: (28+20) AGCRAAAGCAGGTAGATGTTGAAAGATGAAAACTACCTTGTTTCT ACT 111) SEQ ID NO: 443 BM18_M, 1, 1006, 50: (28+22) AGCRAAAGCAGGTAGATGTTGAAAGATGAAAAAACTACCTTGTTT CTACT 112) SEQ ID NO: 444 BM18_M, 1, 1005, 49: (26+23) AGCRAAAGCAGGTAGATGTTGAAAGATAAAAAACTACCTTGTTTC TACT 113) SEQ ID NO: 445 BM18_M, 1, 1010, 43: (25+18) AGCRAAAGCAGGTAGATGTTGAAAGAACTACCTTGTTTCTACT 114) SEQ ID NO: 446 BM18_M, 1, 1006, 47: (25+22) AGCRAAAGCAGGTAGATGTTGAAAGAAAAAACTACCTTGTTTC TACT 115) SEQ ID NO: 447 BM18_M, 1, 1007, 42: (21+21) AGCRAAAGCAGGTAGATGTTGAAAAACTACCTTGTTTCTACT 116) SEQ ID NO: 448 BM18_M, 1, 1002, 46: (20+26) AGCRAAAGCAGGTAGATGTTGAGTAAAAAACTACCTTGTTTC TACT 117) SEQ ID NO: 449 BM18_M, 1, 1003, 44: (19+25) AGCRAAAGCAGGTAGATGTAGTAAAAAACTACCTTGTTTCTACT 118) SEQ ID NO: 450 BM18_M, 1, 1000, 45: (17+28) AGCRAAAGCAGGTAGATTGGAGTAAAAAACTACCTTGTTTCTACT 119) SEQ ID NO: 451 BM18_M, 1, 1005, 40: (17+23) AGCRAAAGCAGGTAGATTAAAAAACTACCTTGTTTCTACT 120) SEQ ID NO: 452 BM18_NA, 1, 1426, 64: (32+32) AGCRAAAGCAGGAGTTTAAATGAATCCAAATCGTAGTTTGTTCAA AAAACTCCTTGTTTCTACT 121) SEQ ID NO: 453 BM18_NA, 1, 1418, 63: (23+40) AGCRAAAGCAGGAGTTTAAATGAATTGACAAGTAGTTTGTTCAAA AAACTCCTTGTTTCTACT 122) SEQ ID NO: 454 BM18_NA, 1, 1420, 58: (20+38) AGCRAAAGCAGGAGTTTAAATGACAAGTAGTTTGTTCAAAAAACTC CTTGTTTCTACT 123) SEQ ID NO: 455 BM18_NA, 1, 1421, 55: (18+37) AGCRAAAGCAGGAGTTTAGACAAGTAGTTTGTTCAAAAAACTCCTT GTTTCTACT 124) SEQ ID NO: 456 BM18_NA, 1, 1438, 37: (17+20) AGCRAAAGCAGGAGTTTAAAAACTCCTTGTTTCTACT 125) SEQ ID NO: 457 BM18_NP, 1, 1548, 57: (39+18) AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAATAC CCTTGTTTCTACT 126) SEQ ID NO: 458 BM18_NP, 1, 1545, 60: (39+21) AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAAAAT ACCCTTGTTTCTACT 127) SEQ ID NO: 459 BM18_NP, 1, 1547, 57: (38+19) AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATAAATACCC TTGTTTCTACT 128) SEQ ID NO: 460 BM18_NP, 1, 1535, 67: (36+31) AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACACAATTAAAG AAAAATACCCTTGTTTCTACT 129) SEQ ID NO: 461 BM18_NP, 1, 1548, 39: (21+18) AGCRAAAGCAGGGTAGATAATAATACCCTTGTTTCTACT 130) SEQ ID NO: 462 BM18_NP, 1, 1542, 42: (18+24) AGCRAAAGCAGGGTAGATAAGAAAAATACCCTTGTTTCTACT 131) SEQ ID NO: 463 BM18_NP, 1, 1537, 47: (18+29) AGCRAAAGCAGGGTAGATAATTAAAGAAAAATACCCTTGTTTCT ACT 132) SEQ ID NO: 464 BM18_NP, 1, 1535, 49: (18+31) AGCRAAAGCAGGGTAGATACAATTAAAGAAAAATACCCTTGTTTC TACT 133) SEQ ID NO: 465 BM18_NP, 1, 1520, 62: (16+46) AGCRAAAGCAGGGTAGATGCAGAGGAGTACGACAATTAAAGAAAAA TACCCTTGTTTCTACT 134) SEQ ID NO: 466 BM18_PA, 1, 2216, 56: (38+18) AGCRAAAGCAGGTACTGATTCAAAATGGAAGACTTTGTAAGTACCTT GTTTCTACT 135) SEQ ID NO: 467 BM18_PA, 1, 2216, 54: (36+18) AGCRAAAGCAGGTACTGATTCAAAATGGAAGACTTTAAGTACCTTGT TTCTACT 136) SEQ ID NO: 468 BM18_PA, 1, 2198, 61: (25+36) AGCRAAAGCAGGTACTGATTCAAAATATCCATACTGTCCAAAAAAGT ACCTTGTTTCTACT 137) SEQ ID NO: 469 BM18_PA, 1, 2215, 40: (21+19) AGCRAAAGCAGGTACTGATTCAAAGTACCTTGTTTCTACT 138) SEQ ID NO: 470 BM18_PA, 1, 2209, 43: (18+25) AGCRAAAGCAGGTACTGATCCAAAAAAGTACCTTGTTTCTACT 139) SEQ ID NO: 471 BM18_PA, 1, 2212, 39: (17+22) AGCRAAAGCAGGTACTGAAAAAAGTACCTTGTTTCTACT 140) SEQ ID NO: 472 BM18_PB2, 1, 2312, 64: (34+30) AGCRAAAGCAGGTCAATTATATTCAATATGGAAAGAATAGTTTAA AAACGACCTTGTTTCTACT 141) SEQ ID NO: 473 BM18_PB2, 1, 2313, 56: (27+29) AGCRAAAGCAGGTCAATTATATTCAATAATAGTTTAAAAACGACCT TGTTTCTACT 142) SEQ ID NO: 474 BM18_PB2, 1, 2320, 48: (26+22) AGCRAAAGCAGGTCAATTATATTCAATAAAAACGACCTTGTTTCTACT 143) SEQ ID NO: 475 BM18_PB2, 1, 2305, 63: (26+37) AGCRAAAGCAGGTCAATTATATTCAATAGTGTCGAATAGTTTAAAAA CGACCTTGTTTCTACT 144) SEQ ID NO: 476 BM18_PB2, 1, 2321, 45: (24+21) AGCRAAAGCAGGTCAATTATATTCAAAAACGACCTTGTTTCTACT 145) SEQ ID NO: 477 BM18_PB2, 1, 2316, 50: (24+26) AGCRAAAGCAGGTCAATTATATTCAGTTTAAAAACGACCTTGTTT CTACT 146) SEQ ID NO: 478 BM18_PB2, 1, 2320, 41: (19+22) AGCRAAAGCAGGTCAATTATAAAAACGACCTTGTTTCTACT 147) SEQ ID NO: 479 BM18_PB2, 1, 2293, 67: (18+49) AGCRAAAGCAGGTCAATTATGGCCATCAATTAGTGTCGAATAGTTTAAA AACGACCTTGTTTCTACT 148) SEQ ID NO: 480 BM18_PB2, 1, 2324, 35: (17+18) AGCRAAAGCAGGTCAATAACGACCTTGTTTCTACT 149) SEQ ID NO: 481 BM18_PB1, 1, 2319, 75: (52+23) AGCRAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACTT TTCTTGAAAAAATGCCTTGTTTCTACT 150) SEQ ID NO: 482 BM18_PB1, 1, 2324, 62: (44+18) AGCRAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTAAAT GCCTTGTTTCTACT 151) SEQ ID NO: 483 BM18_PB1, 1, 2309, 75: (42+33) AGCRAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTT GTCCTTCATGAAAAAATGCCTTGTTTCTACT 152) SEQ ID NO: 484 BM18_PB1, 1, 2324, 48: (30+18) AGCRAAAGCAGGCAAACCATTTGAATGGATAAATGCCTTGTTT CTACT 153) SEQ ID NO: 485 BM18_PB1, 1, 2322, 50: (30+20) AGCRAAAGCAGGCAAACCATTTGAATGGATAAAAATGCCTTGTTT CTACT 154) SEQ ID NO: 486 BM18_PB1, 1, 2318, 50: (26+24) AGCRAAAGCAGGCAAACCATTTGAATATGAAAAAATGCCTTGTTT CTACT 155) SEQ ID NO: 487 BM18_PB1, 1, 2319, 45: (22+23) AGCRAAAGCAGGCAAACCATTTTGAAAAAATGCCTTGTTTCTACT 156) SEQ ID NO: 488 BM18_PB1, 1, 2299, 64: (21+43) AGCRAAAGCAGGCAAACCATTTGAATTTAGCTTGTCCTTCATGAA AAAATGCCTTGTTTCTACT 157) SEQ ID NO: 489 BM18_PB1, 1, 2309, 53: (20+33) AGCRAAAGCAGGCAAACCATTTGTCCTTCATGAAAAAATGCCTTG TTTCTACT 158) SEQ ID NO: 490 BM18_PB1, 1, 2320, 40: (18+22) AGCRAAAGCAGGCAAACCGAAAAAATGCCTTGTTTCTACT 159) SEQ ID NO: 491 BM18_PB1, 1, 2311, 49: (18+31) AGCRAAAGCAGGCAAACCGTCCTTCATGAAAAAATGCCTTGTTT CTACT 160) SEQ ID NO: 492 BM18_PB1, 1, 2297, 63: (18+45) AGCRAAAGCAGGCAAACCAGTGAATTTAGCTTGTCCTTCATGAA AAAATGCCTTGTTTCTACT 161) SEQ ID NO: 493 BM18_PB1, 1, 2317, 42: (17+25) AGCRAAAGCAGGCAAACCATGAAAAAATGCCTTGTTTCTACT 162) SEQ ID NO: 494 BM18_PB1, 1, 2290, 69: (17+52) AGCRAAAGCAGGCAAACCAAAAGTAGTGAATTTAGCTTGTCCTTCA TGAAAAAATGCCTTGTTTCTACT

1) BM18_PB2, 1, 2325, 35: (18+17) SEQ ID NO: 495 AGCRAAAGCAGGTCAATTACGACCTTGTTTCTACT 2) BM18_M, 1, 1010, 35: (17+18) SEQ ID NO: 496 AGCRAAAGCAGGTAGATAACTACCTTGTTTCTACT 3) BM18_M, 1, 1009, 36: (17+19) SEQ ID NO: 497 AGCRAAAGCAGGTAGATAAACTACCTTGTTTCTACT 4) BM18_PB2, 1, 2324, 36: (18+18) SEQ ID NO: 498 AGCRAAAGCAGGTCAATTAACGACCTTGTTTCTACT 5) BM18 NS, 1, 872, 36: (17+19) SEQ ID NO: 499 AGCRAAAGCAGGGTGACAAACACCCTTGTTTCTACT 6) BM18_NP, 1, 1548, 34: (16+18) SEQ ID NO: 500 AGCRAAAGCAGGGTAGAATACCCTTGTTTCTACT 7) BM18_NP, 1, 1547, 37: (18+19) SEQ ID NO: 501 AGCRAAAGCAGGGTAGATAAATACCCTTGTTTCTACT 8) BM18_NP, 1, 1547, 35: (16+19) SEQ ID NO: 502 AGCRAAAGCAGGGTAGAAATACCCTTGTTTCTACT 9) BM18_HA, 1, 1762, 36: (19+17) SEQ ID NO: 503 AGCRAAAGCAGGGGAAAATACACCCTTGTTTCTACT 10) BM18_NS, 1, 873, 35: (17+18) SEQ ID NO: 504 AGCRAAAGCAGGGTGACAACACCCTTGTTTCTACT 11) BM18_NP, 1, 1545, 39: (18+21) SEQ ID NO: 505 AGCRAAAGCAGGGTAGATAAAAATACCCTTGTTTCTACT 12) BM18_HA, 1, 1752, 44: (17+27) SEQ ID NO: 506 AGCRAAAGCAGGGGAAAATAAGGAAAAACACCCTTGTTTCTACT 13) BM18_PB2, 1, 2323, 37: (18+19) SEQ ID NO: 507 AGCRAAAGCAGGTCAATTAAACGACCTTGTTTCTACT 14) BM18_NS, 1, 871, 37: (17+20) SEQ ID NO: 508 AGCRAAAGCAGGGTGACAAAACACCCTTGTTTCTACT 15) BM18_HA, 1, 1761, 37: (19+18) SEQ ID NO: 509 AGCRAAAGCAGGGGAAAATAACACCCTTGTTTCTACT 16) BM18_NP, 1, 1546, 36: (16+20) SEQ ID NO: 510 AGCRAAAGCAGGGTAGAAAATACCCTTGTTTCTACT 17) BM18_HA, 1, 1757, 41: (19+22) SEQ ID NO: 511 AGCRAAAGCAGGGGAAAATGAAAAACACCCTTGTTTCTACT 18) BM18_NS, 1, 869, 39: (17+22) SEQ ID NO: 512 AGCRAAAGCAGGGTGACAAAAAACACCCTTGTTTCTACT 19) BM18_NP, 1, 1546, 38: (18+20) SEQ ID NO: 513 AGCRAAAGCAGGGTAGATAAAATACCCTTGTTTCTACT 20) BM18_PB2, 1, 2319, 39: (16+23) SEQ ID NO: 514 AGCRAAAGCAGGTCAATTAAAAACGACCTTGTTTCTACT 21) BM18_M, 1, 1008, 37: (17+20) SEQ ID NO: 515 AGCRAAAGCAGGTAGATAAAACTACCTTGTTTCTACT 22) BM18_HA, 1, 1760, 38: (19+19) SEQ ID NO: 516 AGCRAAAGCAGGGGAAAATAAACACCCTTGTTTCTACT 23) BM18_NP, 1, 1540, 43: (17+26) SEQ ID NO: 517 AGCRAAAGCAGGGTAGATAAAGAAAAATACCCTTGTTTCTACT 24) BM18_M, 1, 1005, 39: (16+23) SEQ ID NO: 518 AGCRAAAGCAGGTAGATAAAAAACTACCTTGTTTCTACT 25) BM18_PB1, 1, 2324, 36: (18+18) SEQ ID NO: 519 AGCRAAAGCAGGCAAACCAAATGCCTTGTTTCTACT 26) BM18_NP, 1, 1544, 40: (18+22) SEQ ID NO: 520 AGCRAAAGCAGGGTAGATGAAAAATACCCTTGTTTCTACT 27) BM18_NP, 1, 1549, 35: (18+17) SEQ ID NO: 521 AGCRAAAGCAGGGTAGATATACCCTTGTTTCTACT 28) BM18_M, 1, 1011, 34: (17+17) SEQ ID NO: 522 AGCRAAAGCAGGTAGATACTACCTTGTTTCTACT 29) BM18_PB1, 1, 2323, 37: (18+19) SEQ ID NO: 523 AGCRAAAGCAGGCAAACCAAAATGCCTTGTTTCTACT 30) BM18_HA, 1, 1758, 37: (16+21) SEQ ID NO: 524 AGCRAAAGCAGGGGAAAAAAACACCCTTGTTTCTACT 31) BM18_NP, 1, 1543, 41: (18+23) SEQ ID NO: 525 AGCRAAAGCAGGGTAGATAGAAAAATACCCTTGTTTCTACT 32) BM18_NS, 1, 874, 34: (17+17) SEQ ID NO: 526 AGCRAAAGCAGGGTGACACACCCTTGTTTCTACT 33) BM18_PA, 1, 2216, 34: (16+18) SEQ ID NO: 527 AGCRAAAGCAGGTACTAAGTACCTTGTTTCTACT 34) BM18_HA, 1, 1756, 42: (19+23) SEQ ID NO: 528 AGCRAAAGCAGGGGAAAATGGAAAAACACCCTTGTTTCTACT 35) BM18_PB2, 1, 2315, 44: (17+27) SEQ ID NO: 529 AGCRAAAGCAGGTCAATTAGTTTAAAAACGACCTTGTTTCTACT 36) BM18_NS, 1, 870, 38: (17+21) SEQ ID NO: 530 AGCRAAAGCAGGGTGACAAAAACACCCTTGTTTCTACT 37) BM18_PB2, 1, 2322, 38: (18+20) SEQ ID NO: 531 AGCRAAAGCAGGTCAATTAAAACGACCTTGTTTCTACT 38) BM18_PB1, 1, 2325, 35: (18+17) SEQ ID NO: 532 AGCRAAAGCAGGCAAACCAATGCCTTGTTTCTACT 39) BM18_HA, 1, 1758, 38: (17+21) SEQ ID NO: 533 AGCRAAAGCAGGGGAAAAAAAACACCCTTGTTTCTACT 40) BM18_HA, 1, 1754, 42: (17+25) SEQ ID NO: 534 AGCRAAAGCAGGGGAAAAAGGAAAAACACCCTTGTTTCTACT 41) BM18_HA, 1, 1759, 39: (19+20) SEQ ID NO: 535 AGCRAAAGCAGGGGAAAATAAAACACCCTTGTTTCTACT 42) BM18_NA, 1, 1439, 36: (17+19) SEQ ID NO: 536 AGCRAAAGCAGGAGTTTAAAACTCCTTGTTTCTACT 43) BM18_M, 1, 1007, 38: (17+21) SEQ ID NO: 537 AGCRAAAGCAGGTAGATAAAAACTACCTTGTTTCTACT 44) BM18_NA, 1, 1437, 38: (17+21) AGCRAAAGCAGGAGTTTAAAAAACTCCTTGTTTCTACT SEQ ID NO: 538   45) BM18_PB1, 1, 2321, 39: (18+21) SEQ ID NO: 539 AGCRAAAGCAGGCAAACCAAAAAATGCCTTGTTTCTACT 46) BM18_NS, 1, 867, 47: (23+24) SEQ ID NO: 540 AGCRAAAGCAGGGTGACAAAGACATAAAAAACACCCTTGTTTCTACT 47) BM18_NP, 1, 1532, 62: (28+34) SEQ ID NO: 541 AGCRAAAGCAGGGTAGATAATCACTCACACGACAATTAAAGAAAAATAC CCTTGTTTCTACT 48) BM18_NP, 1, 1541, 41: (16+25) SEQ ID NO: 542 AGCRAAAGCAGGGTAGAAAGAAAAATACCCTTGTTTCTACT 49) BM18_PB1, 1, 2322, 38: (18+20) SEQ ID NO: 543 AGCRAAAGCAGGCAAACCAAAAATGCCTTGTTTCTACT 50) BM18_NP, 1, 1531, 52: (17+35) SEQ ID NO: 544 AGCRAAAGCAGGGTAGATACGACAATTAAAGAAAAATACCCTTGTTTCT ACT 51) BM18_PA, 1, 2212, 48: (26+22) SEQ ID NO: 545 AGCRAAAGCAGGTACTGATTCAAAATAAAAAAGTACCTTGTTTCTACT 52) BM18_HA, 1, 1752, 59: (32+27) SEQ ID NO: 546 AGCRAAAGCAGGGGAAAATAAAAACAACCAAAATAAGGAAAAACACCCT TGTTTCTACT 53) BM18_HA, 1, 1754, 41: (16+25) SEQ ID NO: 547 AGCRAAAGCAGGGGAAAAGGAAAAACACCCTTGTTTCTACT 54) BM18_PA, 1, 2211, 43: (20+23) SEQ ID NO: 548 AGCRAAAGCAGGTACTGATTCAAAAAAGTACCTTGTTTCTACT 55) BM18_HA, 1, 1755, 43: (19+24) SEQ ID NO: 549 AGCRAAAGCAGGGGAAAATAGGAAAAACACCCTTGTTTCTACT 56) BM18_HA, 1, 1737, 60: (18+42) SEQ ID NO: 550 AGCRAAAGCAGGGGAAAATAGGATTTCAGAAATATAAGGAAAAACACCC TTGTTTCTACT 57) BM18_PB2, 1, 2313, 47: (18+29) SEQ ID NO: 551 AGCRAAAGCAGGTCAATTAATAGTTTAAAAACGACCTTGTTTCTACT 58) BM18_NS, 1, 864, 50: (23+27) SEQ ID NO: 552 AGCRAAAGCAGGGTGACAAAGACATAATAAAAAACACCCTTGTTTCTAC T 59) BM18_NP, 1, 1546, 56: (36+20) SEQ ID NO: 553 AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACAAAATACCCTTGT TTCTACT 60) BM18_NP, 1, 1535, 59: (28+31) SEQ ID NO: 554 AGCRAAAGCAGGGTAGATAATCACTCACACAATTAAAGAAAAATACCCT TGTTTCTACT 61) BM18_PA, 1, 2212, 44: (22+22) SEQ ID NO: 555 AGCRAAAGCAGGTACTGATTCAAAAAAAGTACCTTGTTTCTACT 62) BM18_PA, 1, 2215, 40: (21+19) SEQ ID NO: 556 AGCRAAAGCAGGTACTGATTCAAAGTACCTTGTTTCTACT 63) BM18_PA, 1, 2208, 47: (21+26) SEQ ID NO: 557 AGCRAAAGCAGGTACTGATTCGTCCAAAAAAGTACCTTGTTTCTACT 64) BM18_PA, 1, 2215, 35: (16+19) SEQ ID NO: 558 AGCRAAAGCAGGTACTAAAGTACCTTGTTTCTACT 65) BM18_PB2, 1, 2313, 53: (24+29) SEQ ID NO: 559 AGCRAAAGCAGGTCAATTATATTCAATAGTTTAAAAACGACCTTGTTTC TACT 66) BM18_PB2, 1, 2314, 46: (18+28) SEQ ID NO: 560 AGCRAAAGCAGGTCAATTATAGTTTAAAAACGACCTTGTTTCTACT 67) BM18_PB2, 1, 2323, 36: (17+19) SEQ ID NO: 561 AGCRAAAGCAGGTCAATAAACGACCTTGTTTCTACT 68) BM18_HA, 1, 1748, 61: (30+31) SEQ ID NO: 562 AGCRAAAGCAGGGGAAAATAAAAACAACCAAAATATAAGGAAAAACACC CTTGTTTCTACT 69) BM18_HA, 1, 1738, 66: (25+41) SEQ ID NO: 563 AGCRAAAGCAGGGGAAAATAAAAACAGGATTTCAGAAATATAAGGAAAA ACACCCTTGTTTCTACT 70) BM18_HA, 1, 1758, 39: (18+21) SEQ ID NO: 564 AGCRAAAGCAGGGGAAAAAAAAACACCCTTGTTTCTACT 71) BM18_HA, 1, 1757, 39: (17+22) SEQ ID NO: 565 AGCRAAAGCAGGGGAAAGAAAAACACCCTTGTTTCTACT 72) BM18_HA, 1, 1738, 58: (17+41) SEQ ID NO: 566 AGCRAAAGCAGGGGAAAAGGATTTCAGAAATATAAGGAAAAACACCCTT GTTTCTACT 73) BM18_NS, 1, 866, 55: (30+25) SEQ ID NO: 567 AGCRAAAGCAGGGTGACAAAGACATAATGGAATAAAAAACACCCTTGTT TCTACT 74) BM18_M, 1, 1011, 35: (18+17) SEQ ID NO: 568 AGCRAAAGCAGGTAGATGACTACCTTGTTTCTACT 75) BM18_NA, 1, 1428, 57: (27+30) SEQ ID NO: 569 AGCRAAAGCAGGAGTTTAAATGAATCCAGTTTGTTCAAAAAACTCCTTG TTTCTACT 76) BM18_NA, 1, 1427, 47: (16+31) SEQ ID NO: 570 AGCRAAAGCAGGAGTTTAGTTTGTTCAAAAAACTCCTTGTTTCTACT 77) BM18_NP, 1, 1548, 57: (39+18) SEQ ID NO: 571 AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAATACCCTTG TTTCTACT 78) BM18_NP, 1, 1535, 57: (26+31) SEQ ID NO: 572 AGCRAAAGCAGGGTAGATAATCACTCACAATTAAAGAAAAATACCCTTG TTTCTACT 79) BM18_PA, 1, 2207, 52: (25+27) SEQ ID NO: 573 AGCRAAAGCAGGTACTGATTCAAAATGTCCAAAAAAGTACCTTGTTTCT ACT 80) BM18_PA, 1, 2215, 36: (17+19) SEQ ID NO: 574 AGCRAAAGCAGGTACTGAAAGTACCTTGTTTCTACT 81) BM18_PA, 1, 2214, 36: (16+20) SEQ ID NO: 575 AGCRAAAGCAGGTACTAAAAGTACCTTGTTTCTACT 82) BM18_PB2, 1, 2320, 48: (26+22) SEQ ID NO: 576 AGCRAAAGCAGGTCAATTATATTCAATAAAAACGACCTTGTTTCTACT 83) BM18_PB1, 1, 2321, 43: (22+21) SEQ ID NO: 577 AGCRAAAGCAGGCAAACCATTTAAAAAATGCCTTGTTTCTACT 84) BM18_PB1, 1, 2319, 44: (21+23) SEQ ID NO: 578 AGCRAAAGCAGGCAAACCATTTGAAAAAATGCCTTGTTTCTACT 85) BM18_PB1, 1, 2320, 39: (17+22) SEQ ID NO: 579 AGCRAAAGCAGGCAAACGAAAAAATGCCTTGTTTCTACT 86) BM18_HA, 1, 1752, 45: (18+27) SEQ ID NO: 580 AGCRAAAGCAGGGGAAAAATAAGGAAAAACACCCTTGTTTCTACT 87) BM18_HA, 1, 1731, 66: (18+48) SEQ ID NO: 581 AGCRAAAGCAGGGGAAAATGAGATTAGGATTTCAGAAATATAAGGAAAA ACACCCTTGTTTCTACT 88) BM18_NS, 1, 869, 52: (30+22) SEQ ID NO: 582 AGCRAAAGCAGGGTGACAAAGACATAATGGAAAAAACACCCTTGTTTCT ACT 89) BM18_NS, 1, 872, 40: (21+19) SEQ ID NO: 583 AGCRAAAGCAGGGTGACAAAGAAACACCCTTGTTTCTACT 90) BM18_M, 1, 1004, 41: (17+24) SEQ ID NO: 584 AGCRAAAGCAGGTAGATGTAAAAAACTACCTTGTTTCTACT 91) BM18_NA, 1, 1433, 53: (28+25) SEQ ID NO: 585 AGCRAAAGCAGGAGTTTAAATGAATCCAGTTCAAAAAACTCCTTGTTTC TACT 92) BM18_NP, 1, 1544, 61: (39+22) SEQ ID NO: 586 AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCGAAAAATACC CTTGTTTCTACT 93) BM18_NP, 1, 1550, 37: (21+16) SEQ ID NO: 587 AGCRAAAGCAGGGTAGATAATTACCCTTGTTTCTACT 94) BM18_NP, 1, 1529, 55: (18+37) SEQ ID NO: 588 AGCRAAAGCAGGGTAGATAGTACGACAATTAAAGAAAAATACCCTTGTT TCTACT 95) BM18_NP, 1, 1544, 38: (16+22) SEQ ID NO: 589 AGCRAAAGCAGGGTAGGAAAAATACCCTTGTTTCTACT 96) BM18_PA, 1, 2207, 64: (37+27) SEQ ID NO: 590 AGCRAAAGCAGGTACTGATTCAAAATGGAAGACTTTGTGTCCAAAAAAG TACCTTGTTTCTACT 97) BM18_PA, 1, 2214, 41: (21+20) SEQ ID NO: 591 AGCRAAAGCAGGTACTGATTCAAAAGTACCTTGTTTCTACT 98) BM18_PA, 1, 2217, 33: (16+17) SEQ ID NO: 592 AGCRAAAGCAGGTACTAGTACCTTGTTTCTACT 99) BM18_PB2, 1, 2324, 35: (17+18) SEQ ID NO: 593 AGCRAAAGCAGGTCAATAACGACCTTGTTTCTACT 100) BM18_PB1, 1, 2294, 74: (26+48) SEQ ID NO: 594 AGCRAAAGCAGGCAAACCATTTGAATAGTAGTGAATTTAGCTTGTCCTT CATGAAAAAATGCCTTGTTTCTACT 101) BM18_PB1, 1, 2326, 36: (20+16) SEQ ID NO: 595 AGCRAAAGCAGGCAAACCATATGCCTTGTTTCTACT 102) BM18_PB1, 1, 2320, 40: (18+22) SEQ ID NO: 596 AGCRAAAGCAGGCAAACCGAAAAAATGCCTTGTTTCTACT 103) BM18_PB1, 1, 2324, 35: (17+18) SEQ ID NO: 597 AGCRAAAGCAGGCAAACAAATGCCTTGTTTCTACT 104) BM18_PB1, 1, 2323, 36: (17+19) SEQ ID NO: 598 AGCRAAAGCAGGCAAACAAAATGCCTTGTTTCTACT 105) BM18_HA, 1, 1737, 75: (33+42) SEQ ID NO: 599 AGCRAAAGCAGGGGAAAATAAAAACAACCAAAATAGGATTTCAGAAATA TAAGGAAAAACACCCTTGTTTCTACT 106) BM18_HA, 1, 1757, 43: (21+22) SEQ ID NO: 600 AGCRAAAGCAGGGGAAAATAAGAAAAACACCCTTGTTTCTACT 107) BM18_NS, 1, 868, 63: (40+23) SEQ ID NO: 601 AGCRAAAGCAGGGTGACAAAGACATAATGGATTCTAACACTAAAAAACA CCCTTGTTTCTACT 108) BM18_NS, 1, 871, 45: (25+20) SEQ ID NO: 602 AGCRAAAGCAGGGTGACAAAGACATAAAACACCCTTGTTTCTACT 109) BM18_NS, 1, 862, 53: (24+29) SEQ ID NO: 603 AGCRAAAGCAGGGTGACAAAGACATAATAATAAAAAACACCCTTGTTTC TACT 110) BM18_NS, 1, 873, 39: (21+18) SEQ ID NO: 604 AGCRAAAGCAGGGTGACAAAGAACACCCTTGTTTCTACT 111) BM18_NS, 1, 869, 43: (21+22) SEQ ID NO: 605 AGCRAAAGCAGGGTGACAAAGAAAAAACACCCTTGTTTCTACT 112) BM18_NS, 1, 837, 73: (19+54) SEQ ID NO: 606 AGCRAAAGCAGGGTGACAAAAGAACTTTCTCGTTTCAGCTTATTTAATA ATAAAAAACACCCTTGTTTCTACT 113) BM18_NS, 1, 875, 33: (17+16) SEQ ID NO: 607 AGCRAAAGCAGGGTGACCACCCTTGTTTCTACT 114) BM18_M, 1, 1006, 69: (47+22) SEQ ID NO: 608 AGCRAAAGCAGGTAGATGTTGAAAGATGAGTCTTCTAACCGAGGTCGAA AAAACTACCTTGTTTCTACT 115) BM18_M, 1, 1010, 45: (27+18) SEQ ID NO: 609 AGCRAAAGCAGGTAGATGTTGAAAGATAACTACCTTGTTTCTACT 116) BM18_M, 1, 1000, 54: (26+28) SEQ ID NO: 610 AGCRAAAGCAGGTAGATGTTGAAAGATGGAGTAAAAAACTACCTTGTTT CTACT 117) BM18_M, 1, 1002, 43: (17+26) SEQ ID NO: 611 AGCRAAAGCAGGTAGATGAGTAAAAAACTACCTTGTTTCTACT 118) BM18_NA, 1, 1429, 56: (27+29) SEQ ID NO: 612 AGCRAAAGCAGGAGTTTAAATGAATCCGTTTGTTCAAAAAACTCCTTGT TTCTACT 119) BM18_NA, 1, 1438, 36: (16+20) SEQ ID NO: 613 AGCRAAAGCAGGAGTTAAAAACTCCTTGTTTCTACT 120) BM18_NP, 1, 1548, 69: (51+18) SEQ ID NO: 614 AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAAATCATGG CGAATACCCTTGTTTCTACT 121) BM18_NP, 1, 1541, 64: (39+25) SEQ ID NO: 615 AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACATCAAAGAAAAAT ACCCTTGTTTCTACT 122) BM18_NP, 1, 1547, 55: (36+19) SEQ ID NO: 616 AGCRAAAGCAGGGTAGATAATCACTCACTGAGTGACAAATACCCTTGTT TCTACT 123) BM18_NP, 1, 1545, 42: (21+21) SEQ ID NO: 617 AGCRAAAGCAGGGTAGATAATAAAAATACCCTTGTTTCTACT 124) BM18_NP, 1, 1550, 34: (18+16) SEQ ID NO: 618 AGCRAAAGCAGGGTAGATTACCCTTGTTTCTACT 125) BM18_NP, 1, 1542, 42: (18+24) SEQ ID NO: 619 AGCRAAAGCAGGGTAGATAAGAAAAATACCCTTGTTTCTACT 126) BM18_NP, 1, 1537, 47: (18+29) SEQ ID NO: 620 AGCRAAAGCAGGGTAGATAATTAAAGAAAAATACCCTTGTTTCTACT 127) BM18_NP, 1, 1535, 49: (18+31) SEQ ID NO: 621 AGCRAAAGCAGGGTAGATACAATTAAAGAAAAATACCCTTGTTTCTACT 128) BM18_NP, 1, 1539, 43: (16+27) SEQ ID NO: 622 AGCRAAAGCAGGGTAGTTAAAGAAAAATACCCTTGTTTCTACT 129) BM18_PA, 1, 2208, 57: (31+26) SEQ ID NO: 623 AGCRAAAGCAGGTACTGATTCAAAATGGAAGGTCCAAAAAAGTACCTTG TTTCTACT 130) BM18_PA, 1, 2216, 39: (21+18) SEQ ID NO: 624 AGCRAAAGCAGGTACTGATTCAAGTACCTTGTTTCTACT 131) BM18_PA, 1, 2213, 41: (20+21) SEQ ID NO: 625 AGCRAAAGCAGGTACTGATTAAAAAGTACCTTGTTTCTACT 132) BM18_PA, 1, 2217, 34: (17+17) SEQ ID NO: 626 AGCRAAAGCAGGTACTGAGTACCTTGTTTCTACT 133) BM18_PA, 1, 2213, 37: (16+21) SEQ ID NO: 627 AGCRAAAGCAGGTACTAAAAAGTACCTTGTTTCTACT 134) BM18_PB2, 1, 2323, 48: (29+19) SEQ ID NO: 628 AGCRAAAGCAGGTCAATTATATTCAATATAAACGACCTTGTTTCTACT 135) BM18_PB2, 1, 2306, 61: (25+36) AGCRAAAGCAGGTCAATTATATTCAAGTGTCGAATAGTTTAAAAACGAC SEQ ID NO: 629   CTTGTTTCTACT 136) BM18_PB2, 1, 2314, 48: (20+28) SEQ ID NO: 630 AGCRAAAGCAGGTCAATTATATAGTTTAAAAACGACCTTGTTTCTACT 137) BM18_PB2, 1, 2326, 34: (18+16) SEQ ID NO: 631 AGCRAAAGCAGGTCAATTCGACCTTGTTTCTACT 138) BM18_PB2, 1, 2293, 67: (18+49) SEQ ID NO: 632 AGCRAAAGCAGGTCAATTATGGCCATCAATTAGTGTCGAATAGTTTAAA AACGACCTTGTTTCTACT 139) BM18_PB2, 1, 2325, 34: (17+17) SEQ ID NO: 633 AGCRAAAGCAGGTCAATACGACCTTGTTTCTACT 140) BM18_PB2, 1, 2308, 51: (17+34) SEQ ID NO: 634 AGCRAAAGCAGGTCAATTGTCGAATAGTTTAAAAACGACCTTGTTTCTA CT 141) BM18_PB2, 1, 2320, 38: (16+22) SEQ ID NO: 635 AGCRAAAGCAGGTCAATAAAAACGACCTTGTTTCTACT 142) BM18_PB1, 1, 2314, 73: (45+28) SEQ ID NO: 636 AGCRAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTTTACTTC ATGAAAAAATGCCTTGTTTCTACT 143) BM18_PB1, 1, 2323, 49: (30+19) SEQ ID NO: 637 AGCRAAAGCAGGCAAACCATTTGAATGGATAAAATGCCTTGTTTCTACT 144) BM18_PB1, 1, 2321, 47: (26+21) SEQ ID NO: 638 AGCRAAAGCAGGCAAACCATTTGAATAAAAAATGCCTTGTTTCTACT 145) BM18_PB1, 1, 2320, 41: (19+22) SEQ ID NO: 639 AGCRAAAGCAGGCAAACCAGAAAAAATGCCTTGTTTCTACT 146) BM18_PB1, 1, 2325, 34: (17+17) SEQ ID NO: 640 AGCRAAAGCAGGCAAACAATGCCTTGTTTCTACT 147) BM18_PB1, 1, 2317, 42: (17+25) SEQ ID NO: 641 AGCRAAAGCAGGCAAACCATGAAAAAATGCCTTGTTTCTACT

#Influenza virus RNA polymerase simulation script that searches for t-loops and checks up/down stream for intermol bp #Uses Python 3.8, biopython, openpyxl, and Vienna RNA 2.47; side packages were installed with Anaconda3 #By AJ te Velthuis, Sept 2020 import sys sys.path.append(“/users/USER/opt/anaconda3/lib/python3.8/site-packages”) ##input sequence. Paste sequence between quotation marks below. RNA = “PASTE SEQUENCE HERE” Name =“Template” ##output bubbles in txt file f= open(“deltaGvalues.txt”,“w+”) ##specify polymerase properties Footprint = 20 ##specify NP footprint NP = 24 TloopDuplex = 48 Duplex = int(TloopDuplex / 2) ##specify window and other comparisons Uloop = “&” #Use & for co-fold to compute long-range interactions between upstream and downstream sequences. Swindow = 1 #size of sliding window ########################################### ##invert input sequence to start at 3′ end NegRNA = RNA[::−1] Length = len(NegRNA) ########################################## ##polymerase bubble properties Bubble = Footprint + Duplex ##iteration start point of simulation; start at nt 2 otherwise downstream sequence is empty for co-fold i = 1 ##end bubble sequence and add 1, because sequence count starts at 0 End = int((Length − Footprint + 1) / Swindow) ##find polymerase bubble sequence; allow for small 3′ part to emerge, but ##then cap how long the emerging sequence can be by assuming that every 24 nt will be bound by NP ##unclear if NP binds in chunks or progressively. Assume that at least 24 nt are needed based on data from Ortin lab. ##stops 1 nt from end to avoid having no sequence in cofold for i in range(1, End−1):  if i <= NP:   Upstream = i   Downstream = 0  else:   Upstream = NP   Downstream = i−NP  #find upstream and downstream sequence of the bubble for intermolecular folding check  Ahead = NegRNA[Footprint+i:Footprint+NP+i]  Aheadinv = Ahead[::−1]  #currently only takes 1 nt of downstream as 0 gives an error in cofold.  Down = NegRNA[Downstream:i]  Downinv = Down[::−1]  ##find the two duplex ends and invert them so they are 5′ to 3′  if i <= Duplex:   Prime3 = NegRNA[0:Upstream]  else:   Prime3 = NegRNA[i−Duplex:i]  Prime3inv = Prime3[::−1]  if i <= End:   Prime5 = NegRNA[Footprint+i:Bubble+i]  else:   Prime5 = NegRNA[Footprint+i:Length]  Prime5inv = Prime5[::−1]  #compute A/U content of nucleotides in active site. Assume window of 6 before and after active site. Various print options are inactivated, but can be used for checking if script works.  ActiveSite = i + 16  #print(“location of bubble is ”, i+1)  #print(“3 prime end 3′ to 5′ is ”, Prime3)  #print(“5 prime end 5′ to 3′ is ”, Prime5inv)  ActiveSiteSeqUp = NegRNA[ActiveSite−1:ActiveSite+4]  ActiveSiteSeqDown = NegRNA[ActiveSite−5:ActiveSite]  #seq_up_list = list(ActiveSiteSeqUp)  seq_up_list = list(ActiveSiteSeqDown)  at_count_up = seq_up_list.count(“a”) + seq_up_list.count (“t”) + seq_up_list.count(“A”) + seq_up_list.count (“T”) + seq_up_list.count (“u”) + seq_up_list.count (“U”)  at_frac_up = float(at_count_up)/5  total_at_up = 100 * at_frac_up ##saving the A/U content is inactivated below #print(total_at_up)  #f.write(“%f\n” % (total_at_up))  ##Vienna RNA package for RNA structure prediction  import RNA  Test = Prime3 + Uloop + Prime5  NegTest = Test[::−1]  #Output = (“>”+Name+“_polymerase_bubble_number_%d\n” % (i+1))  ##To write bubble sequence to .txt file  #f.write(Output)  #f.write(NegTest)  #f.write(“\r\n”)  #use duplex fold to compute t-loop from Vienna package because it ignores intermol bp  duplex = RNA.duplexfold(Prime5inv, Prime3inv)  #print(“%s\n%s [%6.2f]” % (NegTest, duplex.structure, duplex.energy))  ##print(“%6.2f” % (duplex.energy))  #use cofold from Vienna package to check for bp in sequence upstream and downstream of t-loop. Various options can be checked separately, including just upstream seq, just downstream seq, or both seq  Other = Aheadinv + Uloop + Downinv  #Other = Aheadinv  #Other = Downinv  (ss, mfe_dimer) = RNA.cofold(Other)  #print(“%s\n%s [%6.2f]” % (Other, ss, mfe_dimer))  DDeltaG = duplex.energy − mfe_dimer ##compute DeltaDeltaG  ##check if deltaG of t-loop is lower than deltaG of other structures  if duplex.energy >= mfe_dimer:   DeltaG = mfe_dimer * −1  #elif duplex.energy >= 0:   #DeltaG = 0  else:   #DeltaG = duplex.energy   DeltaG = duplex.energy  #DeltaG = mfe_dimer  #DeltaG = duplex.energy  #print(DeltaG)  #print(DDeltaG)  #print(duplex.energy)  #print(“%s\n%s [ %6.2f ]” % (NegTest, ss, mfe))  ##to write deltaG values to .txt file. Various options are available depending on what is being analyzed.  f.write(“%f\n” % (duplex.energy))  #f.write(“%f\n” % (DeltaG))  #f.write(“%f\n” % (mfe_dimer))  #print(NegTest)  #print(“−−−−−”)  i =+ Swindow #################################################### ##close .txt file that deltaG values were written to f.close( ) print (“Done %s” % (Name))

It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

TABLE 1 Sequences of mvRNA templates used. Internal lab Name reference Sequence 5′ and 3′ (vRNA) SEQ ID NO. NP71.1 GC33 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 4 UAGUUAGGUAGUAUACCUAGUAACUAG UCUACCCUGCUUUUGCU NP71.2 GC50.1 AGUAGAAACAAGGGUAUUUUUUUUAC SEQ ID NO: 5 UAGUCCGGUUGUUUUGGUUGCCACUAG UCUACCCUGCUUUUGCU NP71.3 GC50.2 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 6 UAGUCCGCUUGUAUAGCUUGCCACUAG UCUACCCUGCUUUUGCU NP71.4 GC67 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 7 UAGUCCGGCCGAUAUGGCCGCCACUAG UCUACCCUGCUUUUGCU NP71.5 GC83 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 8 UAGUCCGGCCGCCCCGGCCGCCACUAG UCUACCCUGCUUUUGCU NP71.6 GC50.9 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 9 UAGUCCGGCCGUUUUGGUUGCCACUAG UCUACCCUGCUUUUGCU NP71.7 GC50.3 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 10 UAGUCCGGUUCUUUUGGUUGCCACUAG UCUACCCUGCUUUUGCU NP71.8 GC50.4 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 11 UAGUCCGGUUGCUUUGGUUGCCACUAG UCUACCCUGCUUUUGCU NP47 NP47 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 1 UAGUCUACCCUGCUUUUGCU NP56 NP56 AGUAGAAACAAGGGUAUUUUUCUUUCU SEQ ID NO: 2 CGAGCGUACUAGUCUACCCUGCUUUUG CU NP76 NP76 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 3 UAGUGAUUUCGAUGUCACUCUGUGAGU GAUUAUCUACCCUGCUUUUGCU NP71.10 GC50 13 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 12 UAGUGGCAGCAAAAGCAGGGUAACUAG UCUACCCUGCUUUUGCU NP71.11 GC50 15 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 13 UAGUGGCAGCAAAAGCACCCAUACUAG UCUACCCUGCUUUUGCU NP71.12 GC50 16 AGUAGAAACAAGGGUAUUUUUCUUUAC SEQ ID NO: 14 UAGUGGCUCUAAAAGCACCCAUACUAG UCUACCCUGCUUUUGCU

TABLE 2 Cloned PB1 WSN mvRNAs. Length SEQ ID Name (nt) Sequence 5′ and 3′ (vRNA) NO. PB1 A 57 AGUAGAAACAAGGCAUUUUUUCAUGAA SEQ ID AUCCAUUCAAAUGGUUUGCCUGCUUUC NO: 15 GCU PB1 B 57 AGUAGAAACAAGGCAUUUUUUCAUGAA SEQ ID GGACAUUCAAAUGGUUUGCCUGCUUUC NO: 16 GCU PB1 C 66 AGUAGAAACAAGGCAUUUUUUCAUGAA SEQ ID GGACAAGCUAAACAUUCAAAUGGUUUG NO: 17 CCUGCUUUCGCU PB1 D 67 AGUAGAAACAAGGCAUUUUUUCAUGAA SEQ ID GGACAAGCUAAAUCAUUCAAAUGGUUU NO: 18 GCCUGCUUUCGCU PB1 E 62 AGUAGAAACAAGGCAUUUUAAGUCGGA SEQ ID UUGACAUCCAUUCAAAUGGUUUGCCUG NO: 19 CUUUCGCU PB1 F 64 AGUAGAAACAAGGCAUUUUUUCAGUCG SEQ ID GAUUGACAUCCAUUCAAAUGGUUUGCC NO: 20 UGCUUUCGCU PB1 G 52 AGUAGAAACAAGGCAUUUUUUCAUGCA SEQ ID UUCAAAUGGUUUGCCUGCUUUCGCU NO: 21 PB1 H 60 AGUAGAAACAAGGCAUUUUUUCAUGAA SEQ ID GGACAAGCUAAAUUCAGUUUGCCUGCU NO: 22 UUCGCU PB1 I 40 AGUAGAAACAAGGCAUUUUUUCAGUUU SEQ ID GCCUGCUUUCGCU NO: 23 PB1 J 80 AGUAGAAACAAGGCAUUUUUUCAUGAA SEQ ID GGACAAGCUAAAUUCGGAUUGACAUCC NO: 24 AUUCAAAUGGUUUGCCUGCUUUCGCU

TABLE 3 Cloned mvRNAs based on t-loop analysis. Sequence 5′ and 3′ SEQ ID Name (vRNA) NO. PA66 AGUAGAAACAAGGUACUUUU SEQ ID UUGGACAGUAUGGAUAGCAC NO: 25 AUUUUGAAUCAGUACCUGCU UUCGCU PA60 AGUAGAAACAAGGUACUUUU SEQ ID UUGGACAGUAUGCCAUUUUG NO: 26 AAUCAGUACCUGCUUUCGCU HA61 AGUAGAAACAAGGGUGUUUU SEQ ID UCCUUAUAUUUCUGAAAUCC NO: 27 UAAUCUUCCCCUGCUUUUGC U HA58 AGUAGAAACAAGGGUGUUUU SEQ ID UCCUUAUAUUUCUGAAAUCC NO: 28 UAUUCCCCUGCUUUUGCU HA63 AGUAGAAACAAGGGUGUUUU SEQ ID UCCUUAUAUUUCUGAAAUGU NO: 29 UUUUAUUUUCCCCUGCUUUU GCU HA64 AGUAGAAACAAGGGUGUUUU SEQ ID UCCUUAUAUUUCUGAAAUCC NO: 30 UAAUCUCAUUCCCCUGCUUU UGCU